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

Abstract

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.

Figure 1

Respiration of mitochondria on pyruvate (ETC complex I substrate) in the presence of mitochondria-targeted pesticides and after MB addition (the data are shown as mean ± SD, n = 6). Pesticides, MB and mitochondria (120 μg) were directly added to the oxygraph chamber. MB was added to the concentration of 2 μM. The concentration of the pesticides in oxygraph chamber see in 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.

Figure 2

Hydrogen peroxide production of mitochondria on pyruvate (ETC complex I substrate) in the presence of mitochondria-targeted pesticides and after MB addition (the data are shown as mean ± SD, n = 6). Pesticides, MB and mitochondria were directly added to the cuvette. MB was added to the concentration of 2 μM. The concentration of the pesticides in cuvette see in 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.

Figure 3

MB restored mitochondrial membrane potential on glutamate + pyruvate (ETC complex I substrate) after dissipation by fenazaquin (A), pyridaben (B), tolfenpyrad (C), and hydramethylnon (D). Chlorfenapyr uncoupled mitochondrial respiration on both glutamate + pyruvate (E) and α-glycerophosphate (F); 2,4-DNP, 2,4-dinitrophenol. Pesticides, MB and mitochondria were directly added to the cuvette. MB was added to the concentration of 2 μM. The concentration of the pesticide in cuvette see in Table 1, n = 6.

Figure 4

Effect of pesticides and MB on the mitochondrial respiration on ETC complex I substrates (the data are shown as mean ± SD, n = 6); Pyr pyruvate, Glu glutamate. Pesticides, MB and mitochondria (120 μg) were directly added to the oxygraph chamber. MB was added to the concentration of 2 μM. The concentration of the pesticide in oxygraph chamber see in 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.