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. 2022 Apr 20;12(5):615.
doi: 10.3390/life12050615.

Ingestional Toxicity of Radiation-Dependent Metabolites of the Host Plant for the Pale Grass Blue Butterfly: A Mechanism of Field Effects of Radioactive Pollution in Fukushima

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Ingestional Toxicity of Radiation-Dependent Metabolites of the Host Plant for the Pale Grass Blue Butterfly: A Mechanism of Field Effects of Radioactive Pollution in Fukushima

Akari Morita et al. Life (Basel). .

Abstract

Biological effects of the Fukushima nuclear accident have been reported in various organisms, including the pale grass blue butterfly Zizeeria maha and its host plant Oxalis corniculata. This plant upregulates various secondary metabolites in response to low-dose radiation exposure, which may contribute to the high mortality and abnormality rates of the butterfly in Fukushima. However, this field effect hypothesis has not been experimentally tested. Here, using an artificial diet for larvae, we examined the ingestional toxicity of three radiation-dependent plant metabolites annotated in a previous metabolomic study: lauric acid (a saturated fatty acid), alfuzosin (an adrenergic receptor antagonist), and ikarugamycin (an antibiotic likely from endophytic bacteria). Ingestion of lauric acid or alfuzosin caused a significant decrease in the pupation, eclosion (survival), and normality rates, indicating toxicity of these compounds. Lauric acid made the egg-larval days significantly longer, indicating larval growth retardation. In contrast, ikarugamycin caused a significant increase in the pupation and eclosion rates, probably due to the protection of the diet from fungi and bacteria. These results suggest that at least some of the radiation-dependent plant metabolites, such as lauric acid, contribute to the deleterious effects of radioactive pollution on the butterfly in Fukushima, providing experimental evidence for the field effect hypothesis.

Keywords: Fukushima nuclear accident; Oxalis corniculata; Zizeeria maha; alfuzosin; artificial diet; ikarugamycin; lauric acid; low-dose exposure; plant secondary metabolite; radioactive pollution.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure A1
Figure A1
Peak area values of oxalic acid and lauric acid in the targeted GC–MS analysis [49] (left) and peak area values of alfuzosin-related compound and ikarugamycin in the LC–MS analysis [49] (right). Box plots for lauric acid, alfuzosin, and ikarugamycin are also shown in Sakauchi et al. (2021) [49]. IR (shown in red) and NC (shown in green) indicate irradiated samples (n = 3) and nonirradiated control samples (n = 3), respectively. A single black dot represents the mean value of triplicate of a sample. FC indicates fold change of mean values from nonirradiated to irradiated samples. These plots were produced using MetaboAnalyst [96]. Asterisks indicate levels of statistical significance; *, p < 0.05; **, p < 0.01 (t-test).
Figure A2
Figure A2
Identification of the alfuzosin-related metabolite by HPLC. (a) Leaf extract. A peak at 6.74 min was observed. This is one of the triplicate results. The peak area value here is 224.17. (b) Alfuzosin standard (Sigma–Aldrich) 0.1 ng/mL in methanol. The peak at 6.69 min is attributed to alfuzosin, and the peak at 2.09 min is attributed to an impurity in methanol. The peak area value of alfuzosin, 199.36, is similar to that of the alfuzosin-related compound. (c) Methanol only. A peak at 2.12 min was observed, demonstrating impurity.
Figure 1
Figure 1
Ingredients and performance of artificial diets. (a) Ingredients and their weight percentages. AD-FSW-135 has a reduced leaf content. It also has simplified contents with just three ingredients. (b) Survival rate (eclosion rate). AD-FSW-135 shows a higher survival rate than AD-D [24] and a lower rate than AD-F [24] and AD-FSI-112 [64]. The p-values obtained from the χ2 test are shown. The AD-FSW-135 results were obtained from ten biological repeats (see Supplementary Table S1). (c) Male (top) and female (bottom) forewing size. The p-values obtained from the t-test between the natural diet and AD-FSW-135 are indicated. Both natural diet and AD-FSW-135 results were obtained from ten biological repeats (see Supplementary Table S1).
Figure 2
Figure 2
Results of the toxicity test for lauric acid. Asterisks indicate levels of statistical significance in comparison to the control (0 mg/g); *, p < 0.05; **, p < 0.01. These results were obtained from four biological repeats (see Supplementary Table S1). (a) Pupation rate (green), eclosion rate (brown), and normality rate (red). The p-values obtained from the χ2 test are indicated. The pink vertical broken line indicates a rough position of the estimated concentration of lauric acid in irradiated leaves, 0.063 mg/g. (b) Egg-larval days (blue), pupal days (brown), and immature days (gray). The mean values (±standard deviation) are shown as bar height. The p-values obtained from the t-test are indicated. (c) Male (blue green) and female (pink) forewing size. The mean values (±standard deviation) are shown as bar height. The p-values obtained from the t-test are indicated.
Figure 3
Figure 3
Results of the toxicity test for alfuzosin. Asterisks indicate levels of statistical significance in comparison to the control (0 mg/g); *, p < 0.05; **, p < 0.01; ***, p < 0.001. These results were obtained from three biological repeats (see Supplementary Table S1). (a) Pupation rate (green), eclosion rate (brown), and normality rate (red). The p-values obtained from the χ2 test are indicated. The pink vertical broken line indicates a rough position of the estimated concentration of the alfuzosin-related compound in irradiated leaves, 1.6 ng/g. (b) Egg-larval days (blue), pupal days (brown), and immature days (gray). The mean values (±standard deviation) are shown as bar height. The p-values obtained from the t-test are indicated. (c) Male (blue green) and female (pink) forewing size. The mean values (±standard deviation) are shown as bar height. The p-values obtained from the t-test are indicated.
Figure 4
Figure 4
Results of the toxicity test for ikarugamycin. Asterisks indicate levels of statistical significance in comparison to the control (0 mg/g); *, p < 0.05. These results were obtained from three biological repeats (see Supplementary Table S1). (a) Pupation rate (green), eclosion rate (brown), and normality rate (red). The p-values obtained from the χ2 test are indicated. The pink vertical broken line indicates a rough position of the estimated concentration of ikarugamycin in irradiated leaves, 0.26 ng/g. (b) Egg-larval days (blue), pupal days (brown), and immature days (gray). The mean values (±standard deviation) are shown as bar height. The p-values obtained from the t-test are indicated. (c) Male (blue green) and female (pink) forewing size. The mean values (±standard deviation) are shown as bar height. The p-values obtained from the t-test are indicated.
Figure 5
Figure 5
Comparison of the results of the three compounds. The p-values obtained from the χ2 test between lauric acid and alfuzosin are shown. (a) Eclosion (survival) rate (normalized). (b) Normality rate (normalized).

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