**2. Results**

#### *2.1. Fungal Biomass Yield of Metarhizium sp. in the Presence of Acetamiprid*

Among the tested strains, *M. anisopliae* and *M. robertsii* IM2358 were the best growing species, while *M. brunneum* and *M. robertsii* ARSEF727 grew at a similar medium rate (biomass yield about 6gL−1). The slowest growing species was *M. globosum*. It turned out that acetamiprid did not inhibit growth in any of the tested strains, even at the highest concentration of 50 mg L−<sup>1</sup> (*p* > 0.05) (Figure 1). Literature data also provides information that acetamiprid has no harmful e ffect on conidia germination and production or vegetative growth in *M. anisopliae*—strain E9 (ESALQ/USP) [16]. The lack of toxic effect was also confirmed in the presented study.

#### *2.2. Quantitative Analyses of the Content of Acetamiprid in Metarhizium Fungal Cultures*

In this study, the fungal ability to eliminate acetamiprid added to cultures at concentrations of 5, 25 and 50 mg L−<sup>1</sup> was verified (Figure S1). None of the tested *Metarhizium* sp. showed the highly effective ability to remove acetamiprid from the Czapek Dox culture medium after seven days of incubation. At the concentration of 5 mg <sup>L</sup>−1, a slight elimination capacity was demonstrated for *M. brunneum* and *M. robertsii* IM6519 (the average removal rate reached 29 and 24%, respectively). In a situation where five times more acetamiprid was added to the fungal culture, a loss was observed in the samples of all tested strains. *M. anisopliae*, *M. globosum* and *M. brunneum* removed respectively 31, 25 and 24% of acetamiprid from the culture medium. In the case of the other species, the substrate

elimination was below 20%. In the fungal cultures, where the insecticide concentration was 50 mg L−<sup>1</sup> there was no loss of more than 20% of the insecticide for any species. However, studies of the content of acetamiprid separately in the mycelium and culture medium showed that it was accumulated in the fungal cells.

**Figure 1.** Influence of acetamiprid at concentrations of 5, 25 and 50 mg L−<sup>1</sup> on fungal biomass yield of *Metarhizium* sp. One-way ANOVA was used for investigations of statistical significance. All differences are statistically insignificant (*p* > 0.05).

This insecticide has proved the ability to accumulate, inter alia, in the tissues of plants where, through translocation, it can even move from the roots to the shoots [17,18]. It was found that due to its good solubility in water, acetamiprid has a strong toxic effect on aquatic organisms where it bioaccumulates by sorption mechanisms characteristic for compounds with high polarity [19,20]. Although acetamiprid accumulation ability has been described for several different species, in this paper, we present for the first time that entomopathogens, which are often used interchangeably or alternatively with various insecticides in agriculture, can also accumulate this compound, without metabolizing it inside the cells (during seven days of incubation). Herein, the amount of acetamiprid on the mycelium dry weight (mg g<sup>−</sup>1) was directly proportional to the increasing concentration (*p* < 0.05) (Figure 2). This trend was similar for each tested species while *M. brunneum* exhibited the highest accumulation potential. The rest of the strains showed statistically significant differences in the amounts of acetamiprid accumulated in the cells at the three concentrations used. At the concentration of 25 mg L−<sup>1</sup> in *M. anisopliae* the highest amount of the accumulated insecticide (0.69 mg per g of dry weight) was observed (*p* < 0.05), but it is worth noting that this species had been previously found to have high biomass yield (Figure 1)*. M. robertsii* IM6519, *M. robertsii* IM2358 and *M. globosum* accumulated in the cells' comparable amounts of acetamiprid (approximately 0.36–0.45 mg per g of dry weight, *p* > 0.05). By comparison, 0.12 mg per g of dry weight was found in *M. brunneum* (*p* < 0.05). The situation changed for the acetamiprid concentration of 50 mg L−<sup>1</sup> in the fungal culture and the amount of the toxic substrate accumulated in the mycelium leveled out (Figure 2). The largest quantity was detected for *M. globosum* (1.05 mg per g of dry weight), which was different from the other

species (except *M. anisopliae*, *p* > 0.05). In all *M. robertsii* strains and the *M. anisopliae* strain studied, the acetamiprid amounts determined per g of dry weight were comparable.

**Figure 2.** Accumulation of acetamiprid at concentrations of 5, 25 and 50 mg L−<sup>1</sup> in the mycelium of *Metarhizium* sp. One-way ANOVA and Tukey's test were used for investigations of statistical significance. (**<sup>a</sup>**–**<sup>c</sup>**) *p* < 0.05. Statistically significant differences between samples at individual concentrations within the species. (**a**) Between samples with acetamiprid at concentrations of 5 and 25 mg <sup>L</sup>−1; (**b**) between samples with acetamiprid at concentrations of 5 and 50 mg <sup>L</sup>−1; (**c**) between samples with acetamiprid at concentrations of 25 and 50 mg L−1.

#### *2.3. Analyses of Destruxins in Fungal Cultures of Metarhizium sp.*

According to literature data, *M. anisopliae*, *M. robertsii* and *M. brunneum* species have genes responsible for the production of dtxs [8]. Dtxs in *M. globosum* were also marked in this work, but their content was very low compared to the other species (in the order of 0.002 and 0.004 mg L−<sup>1</sup> for dtx A and dtx B, respectively), so this species was excluded from further analyses. *M. robertsii* ARSEF727 had the lowest content of dtxs in the biotic control compared to the other species. The expression of genes responsible for the production of specific units making up the dtxs structure might not be as high as in the case of the other strains [8]. It cannot be said that the low concentrations of dtxs in *M. robertsii* ARSEF727 and *M. globosum* were caused by the poor growth of these fungi, because their growth rate was similar to that of *M. brunneum* and *M. robertsii* IM6519, which turned out to be the species with the highest content of dtxs in the biotic controls. Interestingly, *M. anisopliae*, in which the synthesis of dtxs has been accurately described, did not turn out to be the best producer [21]. *M. robertsii* IM2358 was also found to have higher levels of dtxs than *M. anisopliae*.

According to literature data, none of the fungal species has the capacity to produce all 39 types of dtxs, but *M. anisopliae* produces the majority of them [8]. In all tested strains, except *M. globosum*, 19 dtxs were determined. Due to the lack of chromatography standards, accurate quantitative analyses were performed only for dtx A and B. Therefore, the amounts of dtx A and B and the other types were described separately. It is worth noting that dtx A and dtx B are the main metabolites and occur in higher concentrations compared to other dtxs [22], which was also confirmed in this work.

It was checked whether, despite the lack of the influence of acetamiprid on the growth of the tested fungi, this insecticide affected the secondary metabolism. As mentioned above, three concentrations of acetamiprid (5, 25 and 50 mg <sup>L</sup>−1) were examined. It turned out that the lowest concentration caused disturbances in the synthesis of the secondary metabolites of *Metarhizium* (*p* < 0.05). The use of higher doses of acetamiprid contributed to a gradual reduction in the amount of detected dtxs. Declines in the content of dtxs A and B were quite proportional (Figure 3). For *M. brunneum* there were no statistically significant differences between the amounts of dtx A at concentrations of 25 and 50 mg L−1. The differences between all the concentrations used, which were determined for dtx B were statistically significant. The amounts of dtx A for *M. robertsii* IM2358 did not differ significantly between the concentrations of 5 and 25 mg <sup>L</sup>−1, and for dtx B between the concentrations of 25 and 50 mg L−1. No statistically significant differences between the concentrations of 25 and 50 mg L−<sup>1</sup> for dtx A and B were found for *M. robertsii* ARSEF727.

The most harmful effect of acetamiprid on the production of dtxs was noted for *M. brunneum,* despite the fact that the growth rate and amounts of dtxs in the biotic sample were similar to those observed for *M. robertsii* IM6519. At the concentration of 5 mg <sup>L</sup>−1, the contents of dtxs A and B were 56.43 and 41.93% lower than in the biotic controls, respectively. The highest dose of the insecticide resulted in a very large reduction in the contents of dtxs A and B to 11.99 and 14.58% of the biotic control, respectively. As mentioned above, *M. brunneum* accumulated in the mycelium the lowest quantities of acetamiprid per g of dry weight (Figure 2). It seems that *M. brunneum* defended itself against the presence of the insecticide in the mycelium. This could have been the reason why acetamiprid so heavily influenced the contents of dtxs in this strain. Dtxs production in *M. robertsii* ARSEF727 and *M. robertsii* IM6519 was also inhibited at the highest concentration of the insecticide and for dtx A the amounts were 90.99 and 72.09% lower than in the biotic control, and for dtx B 82.53 and 73.15%, respectively (Figure 3).

Comparable decreases in the contents of dtxs A and B were observed for *M. anisopliae* and *M. robertsii* IM2358 strains. With the increase in the acetamiprid concentration, the amounts of dtx A were lower by 10.04, 17.33 and 43.05% for *M. robertsii* IM2358 and by 13.10, 33.24 and 43.61% for *M. anisopliae*. It was noticed that the dose of 25 mg L−<sup>1</sup> was more toxic to *M. anisopliae* than to *M. robertsii* IM2358. The concentration of dtx B for *M. anisopliae* decreased in a similar way to the content of dtx A (9.49, 33.05 and 40.03% less than in the biotic control, with the increase in the acetamiprid concentration). For *M. robertsii* IM2358 at the of concentrations 5 and 25 mg L−<sup>1</sup> the decreases were similar to dtx A (7.94 and 19.68% less than in the biotic control, respectively), while at the concentration of 50 mg <sup>L</sup>−1, the content of dtx B decreased by 24.21% (the reduction was almost two-fold smaller than for dtx A).

The levels of the other 17 dtxs, for which chromatographic standards are not available, were estimated based on the chromatographic peak areas. It turned out that the tested species differed in terms of the profile of dtxs, which was confirmed by the PCA (Figure 4). To the best of our knowledge, this kind of analysis had never been done before.

A similar dtxs profile was obtained for *M. anisopliae* and *M. robertsii* IM2358 (Figure 4). These strains differed from the others due to the high levels of dtx B1 and dtx Ed. A superior decrease in the levels of these dtxs and dtx D was observed for *M. robertsii* IM2358 with the acetamiprid concentration of 50 mg <sup>L</sup>−1, hence in the PCA chart, this tested sample was distinguished and shown to migrate towards *M. robertsii* IM6519 (tested samples with acetamiprid at concentrations of 5 and 25 mg <sup>L</sup>−1), in which the levels of these dtxs were also low (Figure 4). The level of dtx DesmA distinguished *M. anisopliae* from *M. robertsii* IM2358, as lower values were obtained for *M. anisopliae*. The level of synthesized dtx A1 was definitely a factor differentiating *M. anisopliae* from the other tested species, because for this strain, the highest level of dtx A1 was determined. Such close proximity of *M. anisopliae* and *M. robertsii* IM2358 on the PCA chart was in line with the presented previously. These species were characterized by a similar growth (Figure 1) and the effect of acetamiprid at concentrations of 5, 25 and 50 mg L−<sup>1</sup> on the decrease in the amounts of dtxs A and B (Figure 3).

**Figure 3.** Effect of acetamiprid at concentrations of 5, 25 and 50 mg L−<sup>1</sup> on the amounts of destruxins A (**A**) and B (**B**) produced by *Metarhizium* species. One-way ANOVA and Tukey's test were used for investigations of statistical significance. **\*** *p* < 0.05. Statistically significant differences between samples with acetamiprid at concentrations of 5, 25 and 50 mg L−<sup>1</sup> and their biotic controls within the species; (a–c) *p* < 0.05—statistically significant differences between samples at individual concentrations within the species. (a) Between samples with acetamiprid at concentrations of 5 and 25 mg <sup>L</sup>−1; (b) between samples with acetamiprid at concentrations of 5 and 50 mg <sup>L</sup>−1; (c) between samples with acetamiprid at concentrations of 25 and 50 mg L−1.

**Figure 4.** Results of principal component analysis (PCA) on the profile of destruxins of *Metarhizium* species in samples without the addition of acetamiprid and with acetamiprid at concentrations of 5, 25 and 50 mg L−1. PC1 against PC2 scores chart (**top**); PC1 against PC2 loadings chart (**bottom**).

The above-mentioned results of the accumulation of acetamiprid in the mycelium showed that *M. brunneum* differed from the other species because of the lowest quantity of the insecticide bound in the fungal cells (Figure 2). It turned out that the dtxs profile of this strain also contributed to its differentiation, particularly to the high levels of dtxs DesmB, B2, DesmB2 and E2CL2 and the relatively low levels of dtxs Ed and D2.

Analysis of the PCA chart was more complicated for the strains *M. robertsii* ARSEF727 and *M. robertsii* IM6519. Their profiles were quite similar; however, it was possible to see differences in the contents of individual dtxs (Figure 4). The differentiating factor for *M. robertsii* ARSEF727 was the high level of dtx Ed1, particularly for the sample with acetamiprid at the concentration of 50 mg <sup>L</sup>−1, where the addition of acetamiprid did not reduce the level of dtx Ed1 more than in the sample with the addition of acetamiprid at the concentration of 25 mg L−1. The high level of dtx E2CL2 slightly differentiated from the biotic sample with *M. robertsii* ARSEF727 from the other samples of this species. Dtx CL was at a high level in *M. robertsii* IM6519, but in the sample with acetamiprid at a concentration of 50 mg <sup>L</sup>−1, it definitely decreased. The close proximity of *M. robertsii* IM6519 (the biotic control and the sample with acetamiprid at a concentration of 50 mg <sup>L</sup>−1) to *M. robertsii* ARSEF727 was due to the high level of dtx A3. However, the low level of this dtx in the tested sample with acetamiprid at a concentration of 25 mg L−<sup>1</sup> in *M. robertsii* ARSEF727 made this strain more similar to *M. robertsii* IM6519 (samples with acetamiprid at concentrations of 5 and 25 mg <sup>L</sup>−1).

#### *2.4. Permeability of the Cell Membrane and the Content of Acetamiprid in Spores and Subcellular Fractions of the M. brunneum*

*M. brunneum* was chosen to conduct an experiment on the effect of acetamiprid on the permeability of biological membranes, due to the greatest inhibition of dtxs production by this compound, even to 88% at the concentration of 50 mg L−<sup>1</sup> (Figure 3). Dtxs are extracellular metabolites; however, their synthesis takes place in fungal cells [23]. The properties of the cell membrane, including permeability, potential and fluidity, have an influence on the cell secretion process [24]. Acetamiprid did not reduce the permeability of biological membranes at any of the used concentrations (*p* > 0.05). The reduced amount of dtxs determined in the samples with the addition of the toxic insecticide was not associated with a disorder of the membrane permeability system (Table S1).

Due to the high inhibition of dtxs production, it was checked whether acetamiprid accumulated in the spores of *M. brunneum*. Additionally, its amount in the cell wall and other subcellular structures of the fungus was determined. It turned out that acetamiprid accumulated in the spores (0.280 ± 0.05 μg per 10<sup>6</sup> of spores), while in cell fractions almost 6-fold more acetamiprid was detected in the cell wall (27.61 ± 3.75 μg per g of dry weight) than in the other subcellular structures (4.78 ± 0.65 μg per g of dry weight).

#### *2.5. Influence of Acetamiprid, Spores of M. brunneum ARSEF2107 and the Combination of Spores and Acetamiprid on the Mortality of Tenebrio molitor (Mealworm)*

Acetamiprid is used worldwide as an effective insect control agent. The dose of acetamiprid at used in these studies (5, 25 and 50 mg <sup>L</sup>−1) caused mortality of *T. molitor* (Figure 5).

Additionally, the action of the neonicotinoid was compared to the killing properties of *M. brunneum* spores. The obtained results of acetamiprid accumulation in the fungal spores prompted us to determine what effect could be generated by the combination of *M. brunneum* spores with the accumulated acetamiprid and whether such a mixture could be an alternative to using the chemical insecticide alone. The results were surprising because a dose of 25 μg acetamiprid (50 mg <sup>L</sup>−1) caused similar mealworm mortality resulting from a combination of spores with the insecticide accumulated in the amount of 140 ng, i.e., almost 180-fold less. The difference in LT50 was one day, and for the highest dose of acetamiprid this value was determined on the fifth day of testing, and for the combination of spores and insecticide on day six. Acetamiprid at the highest dose acted slightly faster, while at the end of the experiment it turned out that the highest mortality was achieved for the combined action of spores and acetamiprid. Similarly to chemical insecticides, entomopathogens do not kill insects immediately and their lethal effect is delayed even up to 14 days [13]. When a combination of spores and acetamiprid was used, an effect similar to that achieved by the insecticide applied alone was observed. This suggests that when using a combination of spores and acetamiprid, the mechanism of action of the insecticide is can be followed, but due to the accumulation of acetamiprid in the spores, the form of application of the toxic compound has been changed, and therefore, it could be used in a smaller amount than in a situation when it is applied as the only insect-killing agent.

**Figure 5.** Viability of *Tenebrio molitor* mealworms treated with acetamiprid (ACET) at the concentrations of 5, 25 and 50 mg <sup>L</sup>−1, with *Metarhizium brunneum* spores and a combination of spores and ACET. Statistical significance was assessed by standard deviations.

In the natural environment, acetamiprid can impair dtxs production, thereby affecting the functioning of *Metarhizium* sp. in their habitat and the fight for an ecological niche. Due to the increased infectivity of *Metarhizium* spores applied with acetamiprid, it is possible that not only pests, but also beneficial arthropod species may be exposed to this toxic action.
