Assessment of Mitochondrial Function in the AmE‐711 Honey Bee Cell Line: Boscalid and Pyraclostrobin Effects

There is a growing concern that chronic exposure to fungicides contributes to negative effects on honey bee development, life span, and behavior. Field and caged‐bee studies have helped to characterize the adverse outcomes (AOs) of environmentally relevant exposures, but linking AOs to molecular/cellular mechanisms of toxicity would benefit from the use of readily controllable, simplified host platforms like cell lines. Our objective was to develop and optimize an in vitro‐based mitochondrial toxicity assay suite using the honey bee as a model pollinator, and the electron transport chain (ETC) modulators boscalid and pyraclostrobin as model fungicides. We measured the effects of short (~30 min) and extended exposures (16–24 h) to boscalid and pyraclostrobin on AmE‐711 honey bee cell viability and mitochondrial function. Short exposure to pyraclostrobin did not affect cell viability, but extended exposure reduced viability in a concentration‐dependent manner (median lethal concentration = 4175 µg/L; ppb). Mitochondrial membrane potential (MMP) was affected by pyraclostrobin in both short (median effect concentration [EC50] = 515 µg/L) and extended exposure (EC50 = 982 µg/L) scenarios. Short exposure to 10 and 1000 µg/L pyraclostrobin resulted in a rapid decrease in the oxygen consumption rate (OCR), approximately 24% reduction by 10 µg/L relative to the baseline OCR, and 64% by 1000 µg/L. Extended exposure to 1000 µg/L pyraclostrobin reduced all respiratory parameters (e.g., spare capacity, coupling efficiency), whereas 1‐ and 10‐µg/L treatments had no significant effects. The viability of AmE‐711 cells, as well as the MMP and cellular respiration were unaffected by short and extended exposures to boscalid. The present study demonstrates that the AmE‐711‐based assessment of viability, MMP, and ETC functionality can provide a time‐ and cost‐effective platform for mitochondrial toxicity screening relevant to bees. Environ Toxicol Chem 2024;43:976–987. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.


INTRODUCTION
Nutrient-rich food that is both affordable and accessible is a global public health priority (Food and Agriculture Association of the United Nations, et al., 2022).Efficient food production can help increase supply and reduce costs for consumers; however, agricultural practices aimed at increasing yields and preserving food quality may sometimes be at odds with the pollination of flowering crops.For example, fungicide applications are often necessary to limit the impact of plant fungal diseases, but accumulating evidence suggests that these chemicals can produce negative effects on honey bees and other pollinators (reviewed in Fisher et al., 2023).Fungicides can be applied multiple times to the same crop, even during the bloom period (Favaro et al., 2019;reviewed in Gierer et al., 2019;Graham et al., 2021).Regardless of whether hives are located near flowering crops, honey bees can be exposed to fungicides while foraging in and around treated fields (David et al., 2016;Frazier et al., 2015) or inside the hive where contaminated pollen and nectar could be stored (David et al., 2016;Mullin et al., 2010).Fungicide exposure within the hive is associated with an increased probability of disruptive queen bee events (e.g., supersedure) and colony mortality (Traynor et al., 2016).Fungicides can also have interactive effects via modulation of responses to symbiotic and pathogenic microorganisms (Al Naggar et al., 2022;Pettis et al., 2013;Yoder et al., 2013) and other chemicals, including pollen phytochemicals, insecticides, spray adjuvants, and beekeeperapplied acaricides (Johnson et al., 2013;Mao et al., 2017;Wade et al., 2019;Walker et al., 2022).The risks that fungicides pose for honey bees are variable and depend on several factors, such as method of application, stage of honey bee development, including behavioral development, and the duration, route, and timing of exposure (Fisher et al., 2022;reviewed in Fisher et al., 2023 andin Rondeau &Raine, 2022).Many fungicides are designed to target fungal mitochondrial complexes needed for energy production (reviewed in Bartlett et al., 2002), but they can also have effects on nontarget organisms (reviewed in Wang et al., 2021), including pollinators.For example, Nicodemo et al. (2020) observed inhibition of respiration and adenosine triphosphate (ATP) synthesis in isolated honey bee mitochondria caused by pyraclostrobin.Moreover, some fungicides affect honey bee mitochondrial function through transcription-level changes in genes associated with oxidative phosphorylation and detoxification (Christen et al., 2019).
Fungicides that target individual complexes of the electron transport chain (ETC) have been detected in beeswax and pollen within honey bee hives.These include complex II (boscalid) and complex III (azoxystrobin, fluoxastrobin, pyraclostrobin, and trifloxystrobin) modulators, as well as the ETC uncoupler fluazinam (Ostiguy et al., 2019).The prevalent use of these fungicides and other agrochemicals on bee-visited crops establishes the need to rapidly screen for mitochondrial effects in honey bees.Highthroughput characterization of molecular targets of chemicals can rapidly advance our understanding of higher order adverse responses, including cellular and organismal ones, and has been at the center of the US National Research Council's vision for toxicity testing in the 21st century (reviewed in Villeneuve et al., 2019).Cultured mammalian cells have proved useful for elucidating mechanisms of toxicity for agrochemicals, including those that disrupt mitochondrial function (Rjiba-Touati, et al., 2022;Tao et al., 2020;van der Stel et al., 2020).Although the availability of cell cultures for bee research is limited, a continuous cell line from Apis mellifera, as well as primary cultures from honey bees and bumblebees (Bombus impatiens and B. terrestris), have been suggested as in vitro systems for toxicology studies (Goblirsch and Adamczyk, 2023;Moffat et al., 2015Moffat et al., , 2016;;Walderdorff et al., 2018;Wilson et al., 2013).To facilitate development of cost-effective, rapid screening we: 1) characterized the responses of a honey bee cell line to a set of well-defined mammalian mitochondrial stressors, and 2) evaluated the applicability to a honey bee cell model of mammalian assays that are good indicators of mitochondrial stress (see van der Stel et al., 2020).A continuous cell line established from honey bee embryonic tissues, AmE-711 (Goblirsch et al., 2013), was used to assess the effects on cell viability, mitochondrial membrane potential (MMP), and cellular respiration parameters.Change in MMP was selected as an endpoint because it can be induced by a variety of chemicals, including fungicides, and has been linked to adverse outcomes (AOs) including alterations of the oxidation-reduction potential, mitochondrial membrane permeability transition, mitochondrial morphology, and cell death (van der Stel et al., 2023).Cellular respiration was selected because it is a proxy for mitochondrial function, and because it constitutes a sensitive endpoint likely to be altered after mitochondrial disruption by fungicides (van der Stel et al., 2020).Cellular oxygen consumption was assessed after brief exposure to a series of well-characterized, potent mitochondrial toxicants commonly deployed in mammalian mitochondrial stress assays (e.g., rotenone, oligomycin).Ordered administration of these mitochondrial toxicants allowed for estimation and investigation of a suite of respiratory parameters that included basal, ATP-dependent, maximum, and proton leak-dependent respiration, as well as uncoupling and spare capacity.
were selected as test chemicals because they have been shown to act as mitochondrial stressors in mammalian cells (d 'Hose et al., 2021;Luz et al., 2018;van der Stel et al., 2020).Boscalid is a Group 7 carboxamide that targets fungal succinate dehydrogenase (SDH)/respiratory complex II (CII; reviewed in Avenot & Michailides, 2010).Pyraclostrobin is a Group 11 strobilurin that targets the quinol outer binding site of fungal cytochrome bc1 located in respiratory complex III (CIII; Bartlett et al., 2002).Due to their effectiveness, quinone outside inhibitors like pyraclostrobin represented >15% of the fungicide market share in 2014 (Howell et al., 2014), and their use is growing (US Geological Survey [USGS], 2021).In the United States, boscalid and pyraclostrobin are applied to almond orchards, as well as other flowering crops that honey bees may visit for pollen and nectar (California Department of Pesticide Regulation, 2023; USGS, 2021).Further reasons for assessing these two chemicals are that boscalid and pyraclostrobin have been detected in honey bees, pollen, honey, and beeswax within the hive (David et al., 2016;reviewed in Johnson et al., 2010;Mullin et al., 2010;Simon-Delso et al., 2017), and that adult honey bees exposed to field-relevant concentrations of the formulated product, Pristine ® , which contains both boscalid and pyraclostrobin as active ingredients, show adverse effects on several metrics of honey bee health, including those that could be related to impaired mitochondrial function (DesJardins et al., 2021;Fisher et al., 2021Fisher et al., , 2022;;Glass et al., 2021).The present study evaluated whether AmE-711-based viability, MMP, and extracellular flux assays, which include analyses of ETC functionality, can provide a high-throughput, cost-effective platform for mitochondrial toxicity screening relevant to honey bees.

Culture of AmE-711 cells
The AmE-711 cell line was obtained, with permission, from the University of Minnesota (Minneapolis, MN, USA).This line is persistently infected with the the honey bee positive-sense, single-stranded RNA virus termed Deformed wing virus (Carrillo-Tripp et al., 2016), a common pathogen of honey bee colonies.Routine maintenance of AmE-711 is accomplished by incubating the cells in a growth medium consisting of Schneider's Insect Medium containing L-glutamine and sodium bicarbonate (Millipore Sigma).The growth medium is supplemented with 10% heat-inactivated fetal bovine serum (FBS; Millipore Sigma).Stocks of AmE-711 cells are cultured in sealed 25 to 75-cm 2 flasks (Corning) and maintained at 32 °C in a nonhumidified incubator.For experiments, cells were transferred from stock flasks to multiwell plates by first exposing the cells to 0.25% trypsinethylenediaminetetraacetic acid (Thermo Fisher Scientific) to facilitate detachment.Sterile 1× Dulbecco's phosphate-buffered saline (dPBS), modified without calcium chloride and magnesium chloride (Millipore Sigma), was used to wash the cell layer prior to adding trypsin.Activity of the protease was halted by adding fresh growth medium, and detached cells were collected from one to several flasks and mixed thoroughly prior to seeding equal numbers of cells into wells.

Chemicals
Boscalid (CAS No. 188425-85-6) and pyraclostrobin (CAS No. 175013-18-0) were purchased from Millipore Sigma.Concentrated stocks were prepared by dissolving each compound in 100% dimethyl sulfoxide (DMSO), and these stocks were then stored at 4 °C (for the viability and MMP assays) or -20 °C (for the mitochondrial stress assay) until used in experiments.

Resazurin-based cell viability assay
The change in AmE-711 cell viability in response to either boscalid or pyraclostrobin exposure was quantified using the resazurin-based dye AlamarBlue HS Cell Viability Reagent (Thermo Fisher Scientific).Wells of 96-well tissue culture-treated black plates with clear flat bottoms (Corning) were seeded with approximately 1.0 × 10 5 cells/well in 100 μL of growth medium supplemented with 10% FBS.After seeding, the plates were wrapped with Parafilm and placed in a sealed polybag to reduce evaporation.The plates were then transferred to a nonhumidified incubator and maintained at 32 °C.On Day 3 after seeding, the growth medium was replaced with 100 μL of medium containing a range of concentrations of each active ingredient.The working concentrations were prepared by making a 1:1000 dilution of active ingredients into Schneider's Insect Medium from a series of concentrated stocks prepared in 100% DMSO.The concentrations of active ingredients used to expose the cells were 0.001, 0.01, 0.1, 1.0, 10, 100, 1000, 10,000, and 100,000 µg/L (ppb).A solvent control was included in the series and consisted of 0.1% DMSO, which was also the final concentration of DMSO for each dilution containing the chemical treatment.Cells were incubated in the presence of each chemical concentration for either a short exposure (up to 0.25 h) or an extended exposure (24 h), after which point, 10 μL of AlamarBlue reagent was added directly to each well.After addition of the dye, the total volume of medium in each well was 110 μL.Cells were incubated in the presence of AlamarBlue reagent for 5 h in the dark at 32 °C, and then analyzed by fluorimetry using a Cytation 5 Cell Imaging Multimode Reader connected to Gen 5 3 Ver.10Software (BioTek).Fluorometric parameters consisted of an excitation wavelength of 560/30 nm and an emission wavelength of 590/30 nm.The experiment was repeated three times for each test chemical and duration of exposure.An experiment consisted of three replicate wells/concentration of active ingredient and three replicate wells of solvent control cells.A unique pool of cells was used for each experiment within the exposure group.A set of wells without cells but containing 0.1% DMSO and exposed to the viability reagent was used for blank subtraction.The amount of fluorescence in each well, recorded as optical density units (ODs), was correlated with the number of living cells, and normalized to the average OD of the untreated control wells and presented as the percentage of viability of control.

MMP assay using JC-1 stain
AmE-711 cells were seeded into 96-well tissue culture-treated black plates with clear flat bottoms (Corning) at an approximate density of 1.0 × 10 5 cells/well and allowed to recover from the subculture process as per the viability assay in the previous paragraph.On Day 3 after seeding, the growth medium was removed, and 100 μL of medium containing a range of concentrations of either boscalid or pyraclostrobin in 0.1% DMSO in Schneider's Insect Medium was added to the wells.The concentrations of boscalid or pyraclostrobin followed the same dilution scheme as that used for the viability assay.Cells were incubated in the presence of each test chemical for either a short exposure (up to 0.25 h) or an extended exposure period (24 h).At the end of the exposure period, 10 μL of 10 μg/mL JC-1 Mitochondrial Membrane Potential Probe (Thermo Fisher Scientific) was added to the wells.JC-1 is a membrane-permeable cationic dye that accumulates in mitochondria.The dye forms reversible aggregates in healthy cells that have a normal MMP and negatively charged mitochondrial matrix (Reers et al., 1995).In unhealthy cells, increased membrane permeability and disruption of the electrochemical gradient make the mitochondrial matrix less negative, inhibiting JC-1 entry and aggregate formation (Sivandzade et al., 2019).The aggregate form can be differentiated from the monomeric form using fluorimetry; aggregates fluoresce red and monomers fluoresce green when excited with the appropriate wavelengths.Wells were incubated in the presence of JC-1 for 2 h at 32 °C in the dark.The plates were then analyzed using a Cytation 5 Cell Imaging Multimode Reader connected to Gen 5 Ver.3.10 Software (BioTek).An excitation wavelength of 475/20 nm and an emission wavelength of 530/15 nm were used for monomer (green) detection, and an excitation wavelength of 535/18 nm and an emission wavelength of 590/18 nm were used for aggregate (red) detection.Mitochondrial depolarization is indicated by a decrease in the red:green fluorescence intensity ratio.Three experiments were conducted for each test chemical and exposure period.Each experiment consisted of six replicate wells/concentration, six replicate wells of untreated control cells, and six wells containing culture medium only but stained with JC-1 dye for background subtraction.The ratio of aggregate (red) to monomer (green) signals for each sample was divided by the average red:green signal ratio of the untreated controls and expressed as a percentage of the solvent control.

Mitochondrial stress assay
The Seahorse XFe24 Analyzer (SXA; Agilent Technologies) uses a proprietary technology to measure oxygen concentration rate (OCR), as a proxy of mitochondrial respiration, and extracellular acidification rate (ECAR), as a proxy of glycolysis, from samples consisting of a small amount of tissue, cells, or isolated mitochondria.The multiwell format lends itself to highthroughput assessment of respiration and glycolysis as indicators of cellular metabolic function in real time.To establish whether the AmE-711 cell line could serve as a model system for the identification of chemicals that disrupt mitochondrial function in honey bees, we conducted a set of experiments that used SXA to measure the OCR of cells before and after challenge with a series of known mitochondrial function modulators with welldefined mechanisms of action in mammalian models.The following modulators were used and are listed in the order in which they were administered to cells: 1) oligomycin, 2) carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and 3) rotenone and antimycin-A mixture.Such a sequentially structured chemical challenge also allows for the measurement and calculation of respiration parameters that are indicative of mitochondrial (mal)function.
Boscalid and pyraclostrobin were assessed using two approaches: 1) extended exposure and 2) short exposure.For the extended exposure, cells were incubated with the test chemicals for 16 h prior to the onset of the mitochondrial stress assay.For the short (direct injection) exposure, test chemicals were injected into wells containing cells during the mitochondrial stress assay, which resulted in a 24-min exposure to the test chemicals.Injection of the fungicides occurred after the baseline OCR for the unexposed cells was measured, and before any of the model mitochondrial function modulators were added.
Steps in the SXA assay procedure occurred as follows.On Day 1, the sensor cartridge was immersed in 1000 μL of SXA calibrant fluid for 24 h at 37 °C.The AmE-711 cells were seeded into a 24-well microplate at a seeding density of 50,000 cells/well in 500 μL of culture medium (Schneider's Insect Medium containing L-glutamine, sodium bicarbonate, and 10% FBS).For the extended exposure scenario, cells were combined (on Day 1) with the same culture medium that was enriched with either boscalid or pyraclostrobin at 1, 10, and 1000 µg/L or DMSO (solvent control).The concentration of DMSO was constant across treatments and was below 0.1%.On the morning of Day 2, the assay medium was prepared by combining 500 μL of 100 mmol/L sodium pyruvate, 500 μL of 1 mol/L glucose, and 500 μL of 200 mmol/L glutamine with 48.5 mL of Schneider's medium, adjusted to pH 7.0 to 7.2, and sterilized using a 0.2-µm pore size membrane filter.The culture medium was removed from the cell plate, and the wells were washed twice with 200 μL of assay medium.Each well was refilled with 500 μL of assay medium, and the cell plate was incubated for 1 h at 32 °C to allow the cells to acclimate to the assay medium.
While the cells were acclimating, stock solutions of 100 μmol/L oligomycin, 100 μmol/L of FCCP, and a 50-μmol/L mixture of rotenone and antimycin-A were prepared in assay medium and then diluted further to make working stock solutions that would yield the following concentrations/well: 1 µmol/L oligomycin, 0.5 µmol/L FCCP, and 0.5 µmol/L rotenone and antimycin-A mixture.Mitochondrial modulators were loaded into the injector ports of the sensor cartridge in the order in which they were to be administered into cell wells during the assay: 1) Extended exposure-oligomycin into port A, FCCP into port B, and rotenone and antimycin-A mixture into port C; 2) Short exposure-test chemical or control into port A, oligomycin into port B, FCCP into port C, and rotenone and antimycin-A mixture into port D. After a 1-h incubation, the multiwell plate containing cells and the sensor cartridge containing the mitochondrial modulators and/or test chemicals were placed in the SXA analyzer.The SXA analyzer was programmed to measure the OCR and ECAR three times under basal conditions, and then three times after injection of each test chemical/modulator with each measurement separated by an equal time interval per modulator (24 min).
We ran two separate trials, with separate batches of cells and on different dates, for each chemical by exposure scenario combination, resulting in a total of 10 samples tested/chemical treatment for each of the exposure scenarios.Each trial had its own solvent control wells (0.01% DMSO).Wells that yielded negative spare capacity values were eliminated from the respiratory parameter analyses because their respiratory parameters could not be estimated reliably.This decreased the samples sizes used for respiratory parameter analyses for boscalid to six or seven/treatment for the extended exposure, and five/treatment for the short exposure.For the calculations of the acute change in OCR for the short exposure scenarios, each well had pre-and postchemical injection data collected, and those values were used to calculate the % change in OCR values after chemical addition.

Statistical analyses
Graphical summaries of the data and statistical analyses were carried out using JMP Pro 15.0.0 (JMP Statistical Discovery), Statistica 13.3.(TIBCO Software), or Excel software (Microsoft).For the viability assays, the OD value of each well, including each well of the solvent control, was normalized to the mean OD value of the solvent control wells within each experiment.Viability data are reported as the mean percentage of viability of control ± standard error of the three experiments combined for each active ingredient and exposure period.For the MMP assays, the ratio of the red to green OD values for each well, including each well of the solvent control, was calculated and then normalized to the mean ratio of the red to green OD values of the solvent control wells and expressed as a percentage of the control ± standard error of wells from the three experiments combined for each active ingredient and exposure period.When a dose response was observed, the 10% and 50% lethal concentrations (LC10 and LC50; viability assays) and 10% and 50% effect concentrations (EC10 and EC50; MMP assays) were estimated using a fourparameter logistic model.Normalized data were plotted against the log10-transformed concentration series (µg/L).The parameter estimates with 95% confidence intervals (CIs) were determined.The custom inverse prediction tool in JMP Pro was used to estimate the LC and EC values.Because the mitochondrial stress assay data violated the homogeneity of variance assumption (Levene's test, p > 0.05) for analysis of variance (ANOVA), a nonparametric Kruskal-Wallis one-way ANOVA was used.If a significant treatment effect was found, a post hoc comparison of the mean ranks was conducted following Siegel and Castellan (1988).Excel worksheets containing data used for the generation of the figures and the table in the Results are provided as Supporting Information.

Resazurin-based cell viability assay: Pyraclostrobin
The AmE-711 honey bee cells that were exposed to pyraclostrobin for an extended, but not short, exposure period showed reduced cell viability at the higher end of the concentrations tested (Figure 1).For the short exposure, the mean cell viability remained largely unchanged relative to the solvent control for cells exposed to 0.001 to 100,000 µg/L pyraclostrobin and did not drop below 98% (Figure 1A and Supporting Information, Figure S1A).For the extended exposure period, the mean viability for cells exposed to 0.001 to 100 µg/L was ≥96%.A reduction in the mean viability was apparent for cells exposed to ≥1000 µg/L pyraclostrobin for an extended period, with reductions of 10%, 74%, and 84% for cells exposed to 1000, 10,000, and 100,000 µg/L, respectively (Figure 1B).The LC10 and LC50 for cell viability for extended exposure to pyraclostrobin were determined using a four-parameter logistic model (Supporting Information, Figure S1B).The model incorporated the cell viability data for 10 to 100,000 µg/L to limit the influence of lower concentrations at the upper plateau of the curve that did not produce a response.Results from the model showed a strong negative relationship between cell viability and pyraclostrobin concentration (R 2 = 0.95), yielding an LC10 estimate of 1016 µg/L with 95% CI [650, 1588 µg/L] and an LC50 estimate of 4175 µg/L with 95% CI [3074, 5671 µg/L].
MMP assay using JC-1 stain: Pyraclostrobin Exposure of AmE-711 cells to a range of concentrations of pyraclostrobin (0.001-100,000 µg/L) for both short and extended periods led to a disruption in MMP (Figure 2).Staining of AmE-711 cells with JC-1 dye following both short and extended exposures to pyraclostrobin revealed a dose response suggestive of the MMP disruption.The mean MMP remained relatively unchanged for AmE-711 cells exposed for a short period to 0.001 to 10 µg/L pyraclostrobin (Figure 2A and Supporting Information, Figure S2A).However, the mean MMP showed a steady decline in response to a short exposure to ≥100 µg/L pyraclostrobin.Mitochondrial membrane potential data from the short exposure experiments were input into a logistic model to estimate the EC10 and EC50.Data from concentrations <10 µg/L were excluded from the analyses to diminish the influence of plateau effects on the model (Supporting Information, Figure S2A).A negative relationship between the red:green ratio and pyraclostrobin concentration was observed (R 2 = 0.87), producing an estimated EC10 of 91 µg/L with 95% CI [61, 136 µg/L] and EC50 of 515 µg/L with 95% CI [374, 708 µg/L] for the short exposure.
The MMP showed a pronounced decline over a narrow window of concentrations in response to an extended pyraclostrobin exposure (Figure 2B).Although the mean MMP for AmE-711 cells exposed to 0.001 to 100 µg/L pyraclostrobin appeared to be no different than cells exposed to the solvent control, it decreased rapidly when exposed to ≥1000 µg/L pyraclostrobin.Results from the logistic model (Supporting Information, Figure S2B) showed a negative relationship between the red:green ratio and pyraclostrobin concentration (R 2 = 0.96) and produced an estimated EC10 of 246 µg/L with 95% CI [165,368] and EC50 of 982 µg/L with 95% CI [871, 1107 µg/L].As with the short exposure, data from concentrations <10 µg/L were excluded from the analyses to diminish the influence of plateau effects on the model.

(A) (B)
FIGURE 1: Effects of a short (≤0.25 h; A) and an extended exposure (24 h; B) to pyraclostrobin on honey bee cell viability.Viability was assessed by exposing AmE-711 honey bee cells to a range of concentrations of pyraclostrobin (10 -3 -10 5 µg/L, presented on a log10 scale).Cells were stained with AlamarBlue dye for 5 h after the designated exposure period without removing the active ingredient.Plates were then analyzed by fluorimetry to obtain optical density (OD) readings for each well.Optical density values were blank-subtracted and then normalized to the mean OD value of a set of solvent control wells exposed to 0.1% dimethylsulfoxie and expressed as percentage (%) of control.Response curves were fit to the data points using a four-parameter logistic model.

Mitochondrial stress assay: Pyraclostrobin
Short exposure via injection of 10 and 1000 µg/L pyraclostrobin resulted in a rapid and statistically significant decrease in the OCR (Kruskal-Wallis test, H (3, n = 40) = 32.71,p < 0.05).We observed an approximately 24% reduction by 10 µg/L relative to the baseline OCR, and 64% reduction by 1000 µg/L (Figures 3 and 6).Short exposure to 10 µg/L also significantly decreased coupling efficiency (Table 1), whereas short exposure to 1000 µg/L pyraclostrobin significantly decreased most of the respiratory parameters (i.e., nonmitochondrial, proton-leak associated, maximal and ATP production-associated respiration, and spare capacity and coupling efficiency; Table 1).In the extended exposure scenario, 1000 µg/L pyraclostrobin reduced all respiratory parameters, whereas 1 and 10 µg/L treatments had no significant effects (Table 1 and Supporting Information, Table S1A and B).

Resazurin-based cell viability assay: Boscalid
Exposure of AmE-711 honey bee cells to a wide range of boscalid concentrations (0.001-100,000 µg/L) yielded no effect The ratio of red:green fluorescence intensity for JC-1 staining of AmE-711 honey bee cells after a short (≤0.25 h; A) and an extended exposure (24 h; B) to pyraclostrobin.Red:green fluorescence intensity using JC-1 staining was used as a proxy to assess the change in mitochondrial membrane potential in response to 10 -3 to 10 5 µg/L (presented on a log10 scale) pyraclostrobin at two different exposure periods.Cells were stained with JC-1 for 2 h after exposure to pyraclostrobin, and then red and green signals were quantified using fluorimetry.The mean red:green fluorescence of cells exposed to 0.1% dimethylsulfoxide was used to normalize the ratio of red:green signals for each well exposed to pyraclostrobin and are expressed as the percentage (%) of control.Response curves were fit to the data points using a four-parameter logistic model.
FIGURE 3: Mitochondrial stress assay: Pattern of changes in oxygen consumption rate (pmol O 2 /min) for AmE-711 honey bee cells after direct injection of different concentrations of pyraclostrobin.Tested concentrations were 0 (solvent control) and 10, 100, and 1000 µg/L.Direct injection of pyraclostrobin occurred after the initial three readings to establish basal respiration.A consecutive series of injections with model mitochondrialtoxicants followed pyraclostrobin exposure, and included 1 μM oligomycin, 0.5 μM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, and 0.5 μM rotenone/antimycin-A/mixture.Data are presented as the mean ± standard error.
on mean cell viability after short and extended exposure periods (Figure 4).Relative to cells exposed to the solvent control, the mean cell viability was ≥94% after short (Figure 4A) and extended (Figure 4B) exposures for all boscalid concentrations tested (0.001-100,000 µg/L).Unlike the pyraclostrobin data, cell viability results for boscalid showed that within the range of concentrations tested, the chemical was not toxic to AmE-711 honey bee cells in culture.Due to the lack of effect, LC10 and LC50 values for either short or extended exposures to boscalid were not calculated (Supporting Information, Figure S4A, B).
(A) (B) FIGURE 4: Effects of a short (≤0.25 h; A) and an extended (24 h; B) exposure to boscalid on honey bee cell viability.Viability was assessed by exposing AmE-711 honey bee cells to a range of concentrations of boscalid (10 -3 -10 5 µg/L, presented on a log10 scale).AlamarBlue, a resazurinbased dye, was added to wells after the designated exposure period without removing the active ingredient.The cells were stained with AlamarBlue dye for 5 h prior to fluorimetry.Optical density (OD) readings were blank-subtracted and then normalized to the mean OD value of a set of control wells exposed to 0.01% dimethylsulfoxide and expressed as % of control.Response curves were fit to the data points using a fourparameter logistic model.

MMP assay using JC-1 stain: Boscalid
Exposure of AmE-711 cells to a range of boscalid concentrations for both short and extended periods revealed no clear pattern that would suggest the fungicide had an effect on MMP (Figure 5).The ratio of red:green fluorescence was normalized to the solvent control and expressed as a percentage.Mean ratios ranged from a low of 97% (100,000 µg/L) to a high of 106% (10,000 µg/L) for the short exposure scenario (Figure 5A), and a low of 96% (1 µg/L) to a high of 106% (100,000 µg/L) for the extended exposure scenario (Figure 5B).The mean red: green ratios deviated from the solvent control by <10% for all doses for both exposure periods.The lack of change in MMP response to increasing concentrations of boscalid for either the short or extended exposure periods prevented us from obtaining estimates of either the EC10 or the EC50 (Supporting Information, Figure S5A, B).

Mitochondrial stress assay: Boscalid
Exposure of AmE-711 cells to 1, 10, and 1000 µg/L of boscalid had no effect on any of the respiratory parameters in either the short or extended exposure scenarios (Table 1, Figure 6, and Supporting Information, Table S1C and D).

DISCUSSION
The AmE-711 honey bee cells that were exposed to pyraclostrobin showed a reduction in viability starting at 1000 µg/L (2.6 µM).Similar effect concentrations (7 µM) for pyraclostrobin were reported for HepG2 and RPTEC/TERT1 mammalian cell lines when viability was also assessed with a resazurin reduction assay and cells were exposed for a duration (i.e., 24 h) similar to our extended exposure scenario (van der Stel et al., 2020).In the present study, AmE-711 cells exposed to pyraclostrobin showed a maximum reduction in viability at a concentration of 100,000 µg/L (260 µM); the viability was reduced by 84% after an extended exposure.The LC50 for the short exposure could not be estimated reliably for the range of concentrations we tested; however, the LC50 for cell viability for the extended exposure to pyraclostrobin was 4175 µg/L (10.8 µM).Pyraclostrobin is a mitochondrial toxin developed to target fungal cytochrome bc1 located in respiratory CIII (Avenot & Michailides, 2010;Bartlett et al., 2002).Respiratory CIII facilitates the transfer of electrons to the intermembrane space of mitochondria, creating a proton gradient that drives ATP synthesis (reviewed in Crofts, 2004).Mitochondrial toxin-induced inhibition of CIII can progress to a loss in the electrochemical potential across the inner mitochondrial membrane (see van der Stel et al., 2023).Staining of AmE-711 cells with JC-1 ratiometric dye provided a way for us to quantify whether exposure to pyraclostrobin resulted in alteration of the MMP.There was a decrease in the ratio of JC-1 red:green fluorescence with increasing concentration of pyraclostrobin for both short and extended exposures.This decrease is indicative The ratio of red:green fluorescence intensity for JC-1 staining of AmE-711 honey bee cells after a short (≤0.25 h; A) and an extended exposure (24 h; B) to boscalid.Red:green fluorescence intensity using JC-1 staining was used as a proxy to assess the change in mitochondrial membrane potential in response to 10 -3 to 10 5 µg/L (presented on a log10 scale) boscalid at two different exposure periods.The mean red:green fluorescence of cells exposed to 0.1% dimethylsulfoxide was used to normalize the red:green signal for each well exposed to boscalid and are expressed as the proportion of control.Response curves were fit to the data points using a four-parameter logistic model. of mitochondria that are unable to maintain the electrochemical potential needed for green-fluorescing monomers entering the mitochondrial matrix to form red-fluorescing aggregates, as would occur in healthy cells (Sivandzade et al., 2019).Mammalian RPTEC/TERT1 cells exhibited decreased MMP after a 24-h exposure to 0.4 µM (155 µg/L) pyraclostrobin; the EC50 was 1.78 µM (690 µg/L; van der Stel et al., 2020).These effect concentrations are similar to those generated by the present study with AmE-711 cells with the MMP EC50 being 1.33 µM (515 µg/L) for the short exposure and 2.53 µM (982 µg/L) for the extended exposure.Our EC50 estimates are in the range reported by Nicodemo et al. (2020), who showed a concentration-dependent decrease in the MMP in isolated honey bee mitochondria exposed to 5 or more µM (~2000 µg/L) pyraclostrobin.Given the temporal pattern of onset of MMP, which typically occurs after the initial interactions with ETC complexes (Tebby et al., 2022), we recommend use of the extended exposure scenario (24 h) for assessment of the effects on the MMP.Mitochondrial stress assay-derived OCR responses in AmE-711 cells exposed to known mammalian mitochondrial modulators (i.e., oligomycin, FCCP, rotenone/antimycin-A) were congruent with those reported for mammalian cell models (van der Stel et al., 2020).The AmE-711 cells exposed to oligomycin showed a reduction in OCR.This is consistent with oligomycin's role as a complex V (ATP synthase) inhibitor, and this decrease in OCR could thus be used for estimating the amount of oxygen consumed for mitochondrial ATP production (Divakaruni et al., 2014).Exposure of AmE-711 cells to FCCP increased the mitochondrial respiration rate, which is consistent with the uncoupling role of FCCP in mammalian models.Carbonyl cyanide-4 (triefluoromethoxy) phenylhydrazone uncouples ATP synthesis from oxygen consumption and forces cells into "overdrive" as they attempt to restore MMP.This disturbed, uncoupled state can be used to determine the maximum respiration rate (Divakaruni et al., 2014).The final set of modulators that were administered, rotenone (CI inhibitor) and antimycin-A (CIII inhibitor), inhibit the mitochondrial ETC and can fully shut down mitochondrial respiration (Divakaruni et al., 2014).The pattern of the responses in the AmE-711 cell line to the rotenone/ antimycin-A mixture was equivalent to that observed in mammalian cell lines and was thus suitable for calculating nonmitochondrial respiration.Spare respiratory capacity, which is the difference between the basal and the maximum respiration rates, and proton leak, which is the difference between the basal and the ATP-linked respiration rates, adjusted for nonmitochondrial oxygen consumption, could be readily calculated based on the above parameters following the same protocols suggested for mammalian cells (Agilent Seahorse XFp Cell Mito Stress Test Kit-User Guide Kit 103010-100; Agilent Technologies; Divakaruni et al., 2014).In summary, it is notable that the cell numbers and concentrations of the oligomycin, FCCP, and rotenone/antimycin-A used for AmE-711 in the SXA experiments were well within the range of concentrations suggested for mammalian cells and that they generated qualitatively and quantitatively similar responses to those of mammalian cells (Divakaruni et al., 2014).Initiation of AmE-711 responses by the model mitochondrial modulators did not require cell permeabilization, or sensitization via suppression of glycolysis, but these modifications should be considered because they have been shown to improve the sensitivity of the cellular respiration assays (see van der Stel et al., 2023).
The short-term exposure approach, whereby cells are only briefly exposed to a chemical of interest, was a better choice for SXA mitochondrial respiration/stress screening.Extended exposure of AmE-711 cells to pyraclostrobin resulted in a severe reduction in cell viability, making it more difficult to resolve ETC inhibition-driven, sublethal effects on the OCRs from the cell viability-driven effects.Furthermore, the respiratory outcomes of the short exposure scenario are unlikely to be as affected by compensatory responses as in the extended exposure scenario; this makes interpretation of the data generated by short exposures less complex and better suited for rapid screening of mitochondrial modulation.The present study found that short exposures to 10 and 1000 µg/L (0.026 and 2.6 µM) of pyraclostrobin resulted in 24% and 66% reductions in the basal OCR, respectively.Higher effect concentrations were reported for studies conducted with honey bee mitochondrial isolates.Campbell et al. (2016) tested the effects of Pristine ® , which includes both boscalid and pyraclostrobin, on honey bee mitochondria and reported a decrease in OCRs at ≥5000 µg/L.Nicodemo et al. (2020) tested the effects of pyraclostrobin on oxygen consumption in isolated honey bee mitochondrial protein and observed effects on state III respiration at concentrations ≥10 μM, suggesting that pyraclostrobin has direct effects on the ETC.Mammalian cellbased studies reported effect concentrations similar to the ones we measured for AmE-711 cells; effects on the basal and maximal OCR were reported at 0.4-7 µM for HepG2 and RPTEC/TERT1 cells when they were assessed after injection of pyraclostrobin, which was equivalent to our short exposure (van der Stel et al., 2020).Oxygen consumption rate responses to pyraclostrobin in the AmE-711 system were congruent with pyraclostrobin's proposed mechanism of action as a CIII inhibitor.Complex III inhibitors have been shown to exhibit a dose-dependent decrease in OCR via blocking of the quinol cycle (Q cycle; a mechanism by which CIII moves protons).Further substrate recovery experiments with succinate and ascorbate N,N,Nʹ,Nʹ-tetramethyl-p-phenylenediamine are advised to conclusively demonstrate CIII inhibition potential in the AmE-711 model (see van der Stel et al., 2020).
There were no significant effects of short or extended exposure to boscalid (0.001-100,000 µg/L) on AmE-711 cell viability and MMP.Exposures to 1, 10, and 1000 µg/L showed no effects on any of the respiratory parameters assessed by the mitochondrial stress assay.Our finding of lower sensitivity of AmE-711 cells to a CII inhibitor than to a CIII inhibitor is consistent with findings of van der Stel et al. (2023), who reported that CII inhibitors affected MMP only at the highest concentrations tested and had no effects on cell viability.Some mammalian cell-based studies have reported reduced viability in the range of concentrations tested by the present study.For example, Karakayali et al. (2021) reported reduced viability for the mouse neuroblastoma cell line N2a after 24 to 48-h exposure to boscalid (EC50 = 55.66 μM).d 'Hose et al. (2021) reported an increase in apoptosis in HepG2 cells exposed to 1 μM boscalid for 2 h, but not in peripheral blood mononuclear cells and BJ fibroblasts.The authors suggest that the observed differential sensitivity to boscalid may have been driven by the differences in antioxidant capacity of the cell lines and the differences in mitochondrial content (e.g., hepatoma cells have very high numbers of mitochondria).This explanation was supported by the finding of higher levels of mitochondrial superoxide in HepG2 cells exposed to boscalid (d 'Hose et al., 2021), and is congruent with the findings of Bénit et al. (2019) showing that effects of SDH inhibitors are more pronounced in cell types that are not well equipped to counter oxidative insults, such as cells with impaired superoxide dismutase signaling resulting from defective nuclear factor erythroid 2-related factor 2 translocation.
Low sensitivity to boscalid in our assays could also have been driven by the inherently lower sensitivity of the honey bee mitochondrial respiratory complex to the chemical.This interpretation is supported by the finding that succinate cytochrome c reductase activities in mitochondria isolated from boscalidexposed honey bees had a higher median inhibitory concentration (IC50 value; 76.7 μM) than mitochondria sourced from Homo sapiens (4.8 μM), Lumbricus terrestris (0.36 μM), or Botrytis cinerea (8.6 μM; Bénit et al., 2019).Furthermore, the lack of boscalid's effect on the respiratory parameters could have been caused by the use of nonpermeabilized cells.It has been shown that a variety of CII inhibitors do not alter cell viability, basal OCR, ECAR, or lactate production in living, nonpermeabilized cells at concentrations up to 10 µM (van der Stel et al., 2020).These same authors proposed that the lower toxicity of CII inhibitors could be due to the less critical role of CII in electron transfer and ATP production relative to CI and CIII, and no role in proton pumping.It is notable that the authors point out that a sufficient exposure to CII inhibition would eventually impact the OCR due to nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide depletion (van der Stel et al., 2020).Lastly, it is important to note that the nature of the assay media, which was supplemented with glucose and pyruvate for the mitochondrial stress assay, may have affected our ability to assess CII inhibitors.It has been suggested that the presence of pyruvate in the media can be sufficient to limit the depletion of NADH, via supply to the Krebs cycle up to the succinate oxidation step, and that addition of high levels of glucose can mask the effects of CII inhibitors (Bénit et al., 2019).These outcomes agree with the observation that CII activity is more important for ATP generation under high energy demand (Pfleger et al., 2015).Based on the above, we conclude that there is a need to conduct additional characterization of boscalid, and other CII inhibitors, in a modified assay scenario that would account for these factors, and that further characterization of AmE-711 bioenergetics should be conducted to better optimize the assay for detection of CII inhibitors.
Mitochondrial toxicity data generated using AmE-711 are congruent with the adverse outcome pathway (AOP) framework for mitochondrial ETC inhibition, in which CIII inhibition leads to a decrease in mitochondrial respiration, then to mitochondrial dysfunction typified by changes in MMP, and eventually to cell death (see Tebby et al., 2022).Thus, the AmE-711 system lends itself to quantitative AOP development following the well-developed mammalian cell-based framework described by Tebby et al. (2022).Other wellcharacterized readouts/assays that represent additional key events (KEs) for AOPs can be readily added to the assay battery we have described.Key events are sequentially ordered, causally connected biological events that can result in AOs.Inclusion of additional KEs can help elucidate downstream effects of ETC inhibition, as well as bioenergetic conditions facilitating these responses.For example, the mitochondrial stress assay we conducted with the SXA already incorporates measurement of the ECAR (data not shown), which reflects increased glycolytic activity of cells in response to impaired mitochondrial function (van der Stel et al., 2020).Because the SXA ECAR measurement also includes acidification due to respiratory CO 2 production, glycolytic activity was not quantified based on ECAR in the present study.Instead, we suggest the use of alternative, SXA-compatible glycolytic rate assays that can discern between the two processes.Assays that measure ATP production could also be incorporated.Decreased ATP production due to ETC inhibition has been demonstrated in a honey bee model (Nicodemo et al., 2020) and can provide a linkage to AOs relevant to risk assessment, such as locomotion, thermoregulation, and growth.When one is predicting the effects of ETC inhibition on flight and associated outcomes, a comparison of the sensitivity and metabolic responses of AmE-711 cells and flight muscle cells may be warranted.For example, honey bee flight muscles are an example of extreme adaptation to high energetic needs because they are highly dependent on aerobic metabolism and have mitochondrial densities that reach the theoretical limits of power-generating muscles (Hedges et al., 2019).

CONCLUSIONS
Data generated with this minimal honey bee cell assay battery can be used to rapidly identify and quantify initial mechanistic events associated with mitochondrial modulation by a variety of chemicals, and can inform bee-relevant quantitative AOPs.The results have a potential to initiate adoption of an AmE-711 in vitro platform for high-throughput screening of mitochondrial dysfunction in honey bees and related pollinators.
Supporting Information-The Supporting Information is available on the Wiley Online Library at https://doi.org/10.1002/etc.5847.

FIGURE 6 :
FIGURE 6: Mitochondrial stress assay: acute response.Effects of the short exposure to pyraclostrobin and boscalid on oxygen consumption rate (OCR).Medians, quartiles, and extremes are shown.Statistically significant reduction in OCR was observed in the samples treated with 10 and 1000 µg/L of pyraclostrobin (Kruskal-Wallis analysis of variance with post hoc comparison of the mean ranks; p < 0.05).

TABLE 1 :
Mitochondrial stress assay: Short and extended (16 h) exposure of AmE-711 cells to either boscalid or pyraclostrobin.
Respiratory parameters were calculated based on the oxygen consumption rate (OCR) before and after administration of known mitochondrial modulators.Means (bold) and standard errors (italicized) are shown.*Treatments significantly different from their respective controls (Kruskal-Wallis analysis of variance with post hoc comparison of the mean ranks; p < 0.05, n = 6-10).