Methylene blue can act as an antidote to pesticide poisoning of bumble bee mitochondria

The population of bumble bees and other pollinators has considerably declined worldwide, probably, due to the toxic effect of pesticides used in agriculture. Inexpensive and available antidotes can be one of the solutions for the problem of pesticide toxicity for pollinators. We studied the properties of the thiazine dye Methylene blue (MB) as an antidote against the toxic action of pesticides in the bumble bee mitochondria and found that MB stimulated mitochondrial respiration mediated by Complex I of the electron transport chain (ETC) and increased respiration of the mitochondria treated with mitochondria-targeted (chlorfenapyr, hydramethylnon, pyridaben, tolfenpyrad, and fenazaquin) and non-mitochondrial (deltamethrin, metribuzin, and penconazole) pesticides. MB also restored the mitochondrial membrane potential dissipated by the pesticides affecting the ETC. The mechanism of MB action is most probably related to its ability to shunt electron flow in the mitochondrial ETC.

www.nature.com/scientificreports/ number of such antidotes is very limited. It was found that glucocorticoids and cyclophosphamide significantly alleviate the toxic effects of paraquat, an efficient herbicide used worldwid 53 . Sucralfate 54 and ellagic acid 55 can also prevent the toxicity of paraquat. Lysine acetylsalicylate significantly decreases paraquat toxicity in mammals 56 . Atropine alleviates pesticide poisoning [57][58][59][60] , while oximes act as antidotes against specific pesticides 59,[61][62][63] . Magnesium sulfate can be used for managing the poisoning with organophosphorus pesticides 64 and aluminum phosphide 65 . Pralidoxime and vitamin K are antidotes of organophosphorus insecticides and anticoagulant rodenticides, respectively 66 . 1,8-Naphthalic anhydride is a potential antidote against fungicides 67 . Ozone, both in its gaseous form and dissolved in water, can be used to remove difenoconazole and linuron from carrots 68 .
Methylene blue (MB) is a thiazine dye that has recently attracted a significant attention of researchers because of its newly discovered biological activities. It was found that MB has an antidote effect in methemoglobinemia 69,70 and poisoning with carbon monoxide and cyanide 71,72 . In the mitochondria, MB plays an important role due to its activity as a catalytic redox cycler 73 and can serve as an alternative electron acceptor 74 . MB was found to improve mitochondrial respiration and to decrease oxidative stress in the hearts of diabetic rats 75 , as well as to maintain the function and structure of the retina treated with rotenone (Complex 1 inhibitor) 76 . It also inhibits multiple amine oxidases, thereby preventing chloroacetaldehyde formation. Taking into account the above properties of MB, we believe that MB can be used as an antidote to a wide range of pesticides affecting animals, including pollinators. An important advantage of MB is that it can be added to the syrup fed to bumble bees.
Here, we studied the properties of MB as a potential antidote against the toxic effects of pesticides. To evaluate the protective effect of MB, we measured mitochondrial respiration and membrane potential of the bumble bee mitochondria subjected to the action of various pesticides and treated with MB.

Materials and methods
Bumble bees. B. terrestris (L.) males were provided by the Technology of Bumble Bee Rearing Ltd. (Voronezh, Russia). The bumble bees were kept in cylindrical cages (diameter, 14 cm; height, 7 cm) in the dark at 27-28.5 °C at the air humidity of 55-68%. The bumblebees were fed with 60% inverted sugar syrup.

Isolation of mitochondria.
Bumble bee mitochondria were isolated as described earlier 77 . For each individual experiment, nine B. terrestris males were frozen at − 18 °C for 15 min. The thoraces were separated from the heads and abdomens, placed in 12 ml of ice-cold isolation medium (220 mM mannitol, 100 mM sucrose, 1 mM EGTA, 2 mg/ml fat-free BSA, 20 mM HEPES, pH 7.4) and disintegrated with a 15-ml Dounce tissue grinder. All procedures were performed at 0-4 °C. The homogenate was centrifuged for 5 min at 600 g, and the supernatant was centrifuged for 10 min at 10,000 g. The resulting pellet was resuspended in the washing medium (isolation medium without BSA) and centrifuged for 10 min at 10,000 g. The pellet was resuspended in 100 μl of the washing medium and kept on ice. Isolated mitochondria were used in the experiments within 2 h after isolation. Protein content in the mitochondria was determined with the BCA assay kit (Pierce Biotechnology, USA).
Mitochondrial respiration. The oxygen consumption rate (OCR) in the isolated mitochondria was measured by the amperometric method with a Clark oxygen electrode (Hansatech Instruments, USA). All measurements were performed at 24 °C in 1 ml of incubation medium containing 220 mM mannitol, 100 mM sucrose, 1 mM EGTA, 4 mM potassium phosphate, 20 mM HEPES (pH 7.4), and 5 mM respiratory substrate. MB was added to the concentration of 2 μM (MB concentration was chosen based on the earlier studies of the MB effect on the rat and mouse mitochondria 78,79 . The pesticides were directly added to the oxygraph chamber. The pesticide concentration in the oxygraph chamber was chosen to produce the maximum inhibitory effect (as previously determined for the mitochondrial respiration in vitro, unpublished data) and varied depending on the pesticide ( Table 1). The effect of MB and each of the pesticides on mitochondrial respiration was measured in 6 repetitions (n = 6).
Membrane potential measurements. The membrane potential of the isolated mitochondria was evaluated from changes in the fluorescence of the membrane potential probe Safranin O using a Hitachi F-7000 spectrofluorometer (Hitachi, Japan) 80 at the excitation wavelength of 495 nm and emission wavelength of 586 nm. Incubation medium (1 ml) contained 220 mM mannitol, 100 mM sucrose, 1 mM EGTA, 4 mM potassium phosphate, 0.2 mg/ml BSA, 20 mM HEPES (pH 7.4), 100-120 μg of mitochondrial protein, 2-4 nmol Safranin O, and 10 mM respiratory substrate. MB was added to the concentration of 2 μM. The pesticides were added directly to the cuvette; the pesticide concentration was the same as in the assessment of mitochondrial respiration ( Table 1). The effect of MB and each of the pesticides on mitochondrial membrane potential was measured in 6 repetitions (n = 6). (Sigma, CШA) as described early 81 using a Hitachi F-7000 spectrofluorimeter in 1 ml of the incubation medium (see above) containing 2 μM Amplex Red Ultra, 100-200 μg of mitochondria, and 1 mg/ml horseradish peroxidase (excitation wavelength, 568 nm; emission wavelength, 581 nm). MB was added directly to the cuvette to the concentration of 2 μM. The pesticide concentration was the same as in the assessment of mitochondrial respiration ( Table 1). The effect of MB and each of the pesticides on mitochondrial hydrogen peroxide production was measured in 6 repetitions (n = 6).

Statistical analysis.
Was performed with the STATISTICA software (StatSoft Inc., Tulsa, OK, USA). The results were expressed as mean ± SD. The differences were analyzed with ANOVA and were considered significant at p < 0.05. The effect of MB and each of the pesticides was measured in 6 repetitions (n = 6).

Results
Effect of mitochondria-targeted pesticides and MB on the mitochondria. The respiration of mitochondria from the bumble bee flight muscles on various respiratory substrates (malate, pyruvate, glutamate, proline, succinate, α-glycerophosphate) was measured in the presence and absence of MB. We found that MB stimulated mitochondrial respiration mediated by Complex I on the following respiratory substrates: pyruvate, malate, pyruvate + malate, pyruvate + proline, pyruvate + glutamate. The highest respiratory rate (in the presence of ADP) was observed on pyruvate + glutamate (respiratory control, 6.1); however, the highest respiratory control (14.1) was observed on pyruvate. Thus, the respiratory rate on pyruvate in the absence of MB was 91.51 ± 5.2 nmol O 2 /min mg protein and increased to 115.30 ± 7.21 nmol O 2 /min mg protein after MB addition. At the same time, MB failed to stimulate mitochondrial respiration on succinate and α-glycerophosphate. We also studied the effect of MB on the respiration of mitochondria treated with the mitochondria-targeted pesticides chlorfenapyr, hydramethylnon, pyridaben, tolfenpyrad, and fenazaquin (Sigma-Aldrich, USA).
All mitochondria-targeted pesticides inhibited respiration mediated by Complex I (Fig. 1), which was then restored by the addition of MB.
Next, we estimated the effect of MB and pesticides on the generation of reactive oxygen species (ROS) by the flight muscle mitochondria in vitro. The rate of ROS production was measured on two substrates: pyruvate (respiration mediated by Complex I) and α-glycerophosphate (respiration mediated by the mitochondrial α-glycerophosphate dehydrogenase). We found that MB did not affect the rate of ROS generation by the mitochondria on pyruvate; the production of hydrogen peroxide in the presence MB was 0.9 ± 0.08 nmol H 2 O 2 / min mg protein vs. 0.8 ± 0.10 nmol H 2 O 2 /min mg protein in the absence of MB. Fenazaquin (F(2, 15) = 33.39, p < 0.001), tolfenpyrad (F(2, 15) = 23.39, p < 0.001) and pyridaben (F(2, 15) = 47.31, p < 0.001) have increased the   Table 1. * Statistically significant differences in the mitochondrial respiration rate in the presence of pesticide and pesticide + MB, p < 0.001. ** Statistically significant differences in the mitochondrial respiration rate in the presence of pesticide and pesticide + MB, p < 0.01. *** Statistically significant differences in the mitochondrial respiration rate in the absence and presence of pesticide, p < 0.001.  Table 1. * Statistically significant differences in the hydrogen peroxide production of mitochondria in the absence and presence of pesticide, p < 0.001. ** Statistically significant differences in the hydrogen peroxide production of mitochondria in the presence of pesticide and pesticide + MB, p < 0.01. www.nature.com/scientificreports/ production of hydrogen peroxide on the pyruvate (Fig. 2). No differences in the H 2 O 2 production before and after MB and pesticide addition were found in the mitochondria on α-glycerophosphate (3.1 ± 0.27 H 2 O 2 /min mg protein in the absence of MB vs. 3.5 ± 0.32 H 2 O 2 /min mg protein in the presence of MB). After fenazaquin MB reduced the production of hydrogen peroxide by mitochondria from 1,81 ± 0,23 to 1,38 ± 0,16 nmol H 2 O 2 /min mg protein (Tukey's test, p < 0.01). MB did not reduce or increase production of hydrogen peroxide by mitochondria after other pesticides.
MB restored the mitochondrial membrane potential dissipated by the pesticides affecting the ETC (Fig. 3). Therefore, MB was able to restore both mitochondrial respiration and mitochondrial membrane potential necessary for the ATP production in the mitochondria. We also found that chlorfenapyr uncoupled mitochondrial  www.nature.com/scientificreports/ respiration, since its addition to the mitochondria oxidizing α-glycerophosphate (Fig. 3F) led to the complete loss of membrane potential.

Discussion
Here, we demonstrated that MB stimulates respiration mediated by Complex I of the ETC in the mitochondria from the bumble bee flight muscles. Note that no stimulation of mitochondrial respiration was observed on α-glycerophosphate or succinate, as respiration on these two substrates is not mediated by Complex I. Presumably, MB acts as a redox component of the ETC due to its ability to participate in the NADH oxidation by Complex I and to transfer electrons on cytochrome c, thus providing an alternative electron transfer in the mitochondria.
MB addition caused no statistically significant changes in the rates of ROS production by the mitochondria on α-glycerophosphate and pyruvate, which might be explained by the presumed MB ability to shunt electrons in Complex I, thus ensuring partial reduction of the electron flow through NADH dehydrogenase, which contributes most to the ROS generation.
MB restored respiration in the mitochondria treated with the mitochondria-targeted pesticides, such as pyridaben, chlorfenapyr, fenazaquin, tolfenpyrad, and hydramethylnon. As reported earlier, chlorfenapyr disturbs   Table 1, n = 6. *Statistically significant differences in the mitochondrial respiration rate in the absence and presence of pesticide, p < 0.001. **Statistically significant differences in the mitochondrial respiration rate in the presence of pesticide and pesticide + MB, p < 0.05. www.nature.com/scientificreports/ oxidative phosphorylation in the mitochondria 44 (uncouples oxidative phosphorylation and suppresses ATP production), which might result in the organism death. According to our data, chlorfenapyr inhibited Complex I in the bumble bee flight muscle mitochondria. It also increased the mitochondrial respiration rate on α-glycerophosphate (unpublished data), which was most probably due to the uncoupling effect of this compound. These data confirm our hypothesis that chlorfenapyr uncouples oxidative phosphorylation, as well as inhibits Complex I in the bumble bee flight muscle mitochondria. Hydramethylnon is known to suppress the activity of Complex III of the mitochondrial ETC 41 . We found that that this pesticide indeed inhibited Complex III, because it decreased (1.3 times) the respiration rate on both pyruvate and α-glycerophosphate. However, inhibition by hydramethylnon was more pronounced for the respiration on pyruvate (see above), which suggests that this pesticide inhibited both Complex I and Complex III in the flight muscle mitochondria.
The mechanism of MB action as an antidote might be related to the specific properties of this compound. MB has been long known as an electron carrier 82,83 . It is also a redox mediator capable of oxidizing intramitochondrial NADH and transferring electrons to the downstream components of the ETC. This effect was termed "alternative electron transport" 84 . MB can be reduced by NADH, FADH 2 , and α-glycerophosphate to leucomethylene blue (MBH2), which is then oxidized primarily by cytochrome c 85 . This suggests that MB donates electrons to the Qo ubiquinol-binding site of Complex III.
We have shown earlier that a wide range of pesticides, including non-mitochondrial ones, negatively affect the bioenergetic parameters of mitochondria from the bumble bee flight muscles 86 . Interestingly, MB also stimulated respiration after treatment of the mitochondria with the non-mitochondrial pesticides, such as deltamethrin, metribuzin and penconazole, although to a lesser extent than after the treatment with the mitochondria-targeted pesticides. It is possible that MB can act as an antidote against other (non-mitochondrial) pesticides, but this hypothesis requires further investigation.
The mechanism of the MB-mediated stimulation of respiration in the mitochondria from the bumble bee flight muscles after exposure to pesticides remains unclear. Most probably, the activity of MB is related to its ability to shunt electrons in the mitochondrial ETC. Although further studies are required for the comprehensive understanding of the MB action mechanisms, the obtained results already suggest that MB can be used as an antidote against the toxic action of pesticides in pollinators.

Conclusions
MB stimulated respiration mediated by Complex I in the bumble bee flight muscles mitochondria and restored respiration in the mitochondria treated with the mitochondria-targeted pesticides, such as pyridaben, chlorfenapyr, fenazaquin, tolfenpyrad, and hydramethylnon. MB also stimulated respiration in the mitochondria subjected to the action of non-mitochondrial pesticides, such as deltamethrin, metribuzin and penconazole, although to a lesser extent than in the mitochondria treated with the mitochondria-targeted pesticides. MB restored the mitochondrial membrane potential dissipated by the pesticides affecting the ETC. Taken together, these data demonstrate that MB can be used to reduce the toxicity of pesticides in pollinators. For instance, MB can be added to the syrup used for feeding bumble bees, which might be convenient for the insect treatment in both indoor (greenhouses) and outdoor environments. However, further studies on the effects of MB on bumble bees and other pollinators are needed to elucidate the precise mechanism of action of this antidote.