**3. Conclusions**

The study reveals that, although acetamiprid does not inhibit the growth of *Metarhizium* sp., it affects the metabolism of the fungus by decreasing its ability to produce dtxs. This phenomenon may have environmental implications because dtxs are produced not only during infection but can also be important for the survival of *Metarhizium* in soil. It has been proved for the first time that acetamiprid accumulates in fungi, not only in plant and animal tissues, which could have some ecological implications as well. However, in the case of *Metarhizium*, the most important finding is the fact that acetamiprid increases its infectivity as in the experiments the spores with the accumulated insecticide were shown to cause the highest mortality of the tested larvae. On the other hand, in the light of the results obtained, it cannot be conclusively stated that a decrease in the production of dtxs caused by acetamiprid does not disturb the infection process because acetamiprid is a strong insecticide whose combined action with *M. brunneum* was revealed to be the most effective in the mortality tests of *T. molitor*.

#### **4. Materials and Methods**

#### *4.1. Chemicals and Reagents*

All reagents and solvents were of analytical or liquid chromatography-mass spectrometry (LC–MS) grade and were purchased from Sigma-Aldrich (Steinheim, Germany) unless otherwise stated. Destruxin A (dtx A) from *M. anisopliae* and destruxin B (dtx B; Cayman Chemical, Ann Arbor, MI, USA) were used as chromatography standards. LC–MS grade water (Merck, Darmstadt, Germany) was used for chromatography. Fungi were cultivated on Czapek Dox broth (BD-Difco, Le Pont-de-Claix, France). Acetamiprid (99% purity) was added to the fungal cultures and was also used as a chromatography standard.

#### *4.2. Microorganisms and Cultivation Conditions*

Six fungal strains of the genus *Metarhizium* (*M. robertsii* IM6519, *M. robertsii* IM2358, *M. robertsii* ARSEF727, *M. anisopliae* ARSEF7487, *M. brunneum* ARSEF2107, *M. globosum* ARSEF2596) from the strains collection of the ARSEF (The Agricultural Research Service Collection of Entomopathogenic Fungal Cultures) and the Department of Industrial Microbiology and Biotechnology, University of Lodz (Poland), were used during the investigations. All strains were maintained on ZT agar slants(glucose (4 g <sup>L</sup>−1); Difco yeas<sup>t</sup> extract (4 g <sup>L</sup>−1); agar (25 g <sup>L</sup>−1); and malt extract (6 ◦Blg), up to 1 L; pH 7.0) as described previously [25–27]. Cultures for tests with *Metarhizium* spore suspension adjusted to 1 × 10<sup>6</sup> spores mL−<sup>1</sup> were prepared on Czapek Dox liquid medium (total volume 40 mL in 100 mL Erlenmeyer flasks; BD-Difco, Le Pont-de-Claix, France). Acetamiprid stock dissolved in acetonitrile at the concentration of 50 mg mL−<sup>1</sup> was added to fungal cultures to a final concentration of 5, 25 and 50 mg L−1. Additionally, abiotic controls (without fungal biomass) and biotic controls (without acetamiprid) were prepared. All samples were incubated on a rotary shaker (120 rpm) at 28 ◦C for 7 days.

#### *4.3. Destruxins Extraction by a Modified QuEChERS Method*

After the incubation, the fungal cultures were filtrated through Whatman filter paper Number 1 (Sigma-Aldrich, Steinheim, Germany) to separate the culture media from the mycelia. The mycelia (for biomass estimation) were harvested and dried at 105 ◦C until a constant weight was obtained. The culture media were centrifuged for 5 min at 10,000 rpm, and subsequently, 20 mL was transferred to each 50 mL Falcon tube with 10 mL acetonitrile. After vigorous vortexing for 1 min, 3000 rpm, *QuEChERS* salts (4 g MgSO4; 1 g NaCl; 1 g C6H5 Na3O7·2H2O; 0.5 g C6H6Na2O7·1.5 H2O) were added and the tubes were again vortexed for 1 min. Afterwards, the tubes were centrifuged for 5 min at 8000 rpm. Eight milliliters of the upper layer was transferred to each 15 mL Falcon tube and evaporated to dryness under reduced pressure at 40 ◦C. After evaporation, the samples were dissolved in 5 mL LC–MS grade water (Merck, Darmstadt, Germany) and 4 mL of the extracts were cleaned on the Solid Phase Extraction Column with octadecyl sorbent C18. Subsequently, 5 mL of acetonitrile was added to rinse the metabolites bound on the sorbent, and next, 4 mL of the extract was transferred to each 15 mL Falcon tube and again evaporated to dryness as described above. After evaporation, the samples were dissolved in 2.5 mL of LC–MS grade water and 1 mL was subjected to LC–MS/MS.

#### *4.4. Acetamiprid Extraction by a Modified QuEChERS Method*

The mycelia were separated from Czapek Dox medium by filtration through Whatman filter paper Number 1 (Sigma-Aldrich, Steinheim, Germany). Then, 20 mL of deionized water and 10 mL of acetonitrile were added to each mycelium sample and ultrasonic extraction was done (2 min, Am 36%, pulse for 10 s). Then, 10 mL of acetonitrile was added to the culture medium in the volume 20 mL, and ultrasonic extraction was performed as mentioned above. The subsequent procedure was the same for both types of samples. The samples were transferred to each 50 mL Falcon tube and extracted with a Ball Mill (Retch MM400, Idar-Oberstein, Germany) for 5 min and at 25/s frequency. After homogenization, *QuEChERS salts* were added and the tubes were vortexed 3000 rpm for 1 min. Subsequently, the extracts were centrifuged for 5 min at 5000 rpm. Then, 1 mL of the top layer was collected for the LC–MS/MS analysis.

#### *4.5. Acetamiprid Extraction from M. brunneum Spores and Subcellular Fractions*

To determine the acetamiprid concentration in spores, 7-day-old fungal cultures were filtered through the nylon net. The filtrates were centrifuged for 10 min at 10,000 rpm, and after supernatant removal, 20 mL of deionized water was added to the spores' pellet. The number of spores were counted in the Thoma cell counting chamber. Then, 10 mL of acetonitrile was added and the *QuEChERS* procedure was done as described above.

For acetamiprid presence in cell fractions, fungal cultures were centrifuged (10 min at 10,000 rpm), and after supernatant removal, the precipitate was washed twice with 20 mL of deionized water [28]. Then, the mycelium was suspended in 20 mL of deionized water and ultrasonic extraction was performed (2 min, Am 36%, pulse for 10 s). Mycelia disintegration was controlled by cell oscopic observations. After mycelia disruption, the samples were centrifuged for 10 min at 1200 rpm. The supernatant was separated from the precipitate with the cell wall fraction, then it was transferred into Eppendorf tubes and centrifuged for 20 min at 20,000 rpm at 4 ◦C. Obtained precipitates were suspended in 20 mL of deionized water, then 10 mL of acetonitrile was added and the *QuEChERS* extraction procedure was performed as described above. The cell wall fraction precipitate was washed twice in 20 mL of deionized water by centrifugation for 10 min at 1200 rpm. Then, it was suspended in 20 mL of deionized water, 10 mL of acetonitrile was added and the extraction procedure was followed as described above.

#### *4.6. LC–MS*/*MS Quantitative and Qualitative Analyses of Destruxins*

Quantitative and qualitative analyses of dtxs were carried out by using LC–MS/MS (LC Agilent 1200 coupled with a tandem mass spectrometer, AB Sciex QTRAP 4500, Framingham, MA, USA). The separation was performed with a Kinetex C18 column maintained at 40 ◦C. Water with 5 mM ammonium formate (AF, Solvent A) and methanol with 5 mM ammonium formate (Wolvent B) were used as mobile phases at a flow rate of 0.5 mL min−1. The injection volume was 5 μL. The eluent gradient was conducted as follows: hold 90% A from 0 to 0.25 min, linear increase from 90% A to 90% B to 2 min, hold 90% B from 2 to 4 min, reverse to the initial conditions from 4 to 4.1 min, and maintained for column equilibration to 6.0 min.

The detection of dtxs was conducted using MS/MS with an electrospray ion source (ESI) in the positive ionization scheduled multiple reaction monitoring (sMRM) scan mode. The MRM detection window was set to 25 s. The optimized ESI parameters were as follows: CUR: 25; IS: 5000 V; TEMP: 500 ◦C; GS1: 50; GS2:50. MRM parameters of 19 dtxs are presented in Table S2 [29]. Dtxs were detected in the culture medium of each of the tested strains. The quantitative analyses of dtx A and dtx B were carried out using standard curves in the linearity range 2.5–100 ng mL−<sup>1</sup> (*r* = 0.9997 and *r* = 0.9999, respectively). The levels of the other dtxs were determined based on chromatographic peak areas and compared between samples using principal component analysis (PCA).

#### *4.7. LC–MS*/*MS Quantitative Analyses of Acetamiprid*

Quantitative analyses of acetamiprid were carried out using LC–MS/MS (LC Agilent 1200 coupled with a tandem mass spectrometer, AB Sciex QTRAP 3200). The separation was performed with a Kinetex C18 column maintained at 40 ◦C. Water with 5 mM AF (solvent A) and acetonitrile with 5 mM AF and 0.1% formic acid (FA, Solvent B) were used as mobile phases at a flow rate of 0.5 mL min−1. The injection volume was 10 μL. The eluent gradient was conducted as follows: hold 90% A from 0 to 0.25 min, linear increase from 90% A to 90% B to 0.5 min, hold 90% B from 0.5 to 4 min, reverse to initial conditions from 4 to 4.1 min, and maintained for column equilibration to 6.0 min.

The detection of acetamiprid was conducted using MS/MS with ESI in the positive ionization MRM scan mode (223.2/126.1 *m*/*z*; 223.2/73.0 *m*/*z*). The optimized ESI parameters were as follows: CUR: 25; IS: 5500 V; TEMP: 500 ◦C; GS1: 50; GS2:60. The quantitation curve of acetamiprid was accomplished in the quadratic regression in the range 25–1000 ng mL−<sup>1</sup> and *r* = 0.9999.

#### *4.8. Permeability of the Cell Membranes*

The intention was to check whether acetamiprid simply interferes with the transport of dtxs from the fungal cell to the culture medium, or its action is connected with another mechanism. The procedure was performed according to the method described by Siewiera et al., 2015 [30] with some modifications. Briefly, 1 mL each of the control samples and the tested samples with acetamiprid concentrations of 5, 25 and 50 mg L−<sup>1</sup> was transferred into Eppendorf tubes and then centrifuged for 10 min at 12,000 rpm. The supernatant was removed and 1 mL of Phosphate Buffered Saline (PBS) and 2 μL of propidium iodide (stock solution 0.1 mg mL−1) were added to the precipitate and the mixture was vortexed for 30 s at 3000 rpm. After incubation in the dark for 5 min, the supernatant was removed. The mycelium was washed twice in PBS by centrifugation in the conditions described previously. Finally, the samples were suspended in 1 mL of PBS and propidium iodide fluorescence was measured at λex = 540 and λem = 630 (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany). The final results were presented as fluorescence intensity per mg of dry weight.

#### *4.9. Mortality Test of Larvae of Tenebrio molitor (Mealworm)*

Three concentrations of acetamiprid (5, 25 and 50 mg <sup>L</sup>−1) and *Metarhizium* sp. spores (1 × 10<sup>6</sup> spores mL−1) with and without accumulated acetamiprid were tested. Control samples without any stressful factors were also done. The effects of the individual variants were checked using 10 mealworms, kept in the dark in plastic boxes with holes in the lid, and the bottom was lined with tissue paper. Before starting the experiment, the larvae were fed with oat flakes, which eliminates the death of starvation. Insect vitality was assessed daily for 14 days.
