Phenotypic Plasticity, Epigenetic or Genetic Modifications in Relation to the Duration of Cd-Exposure within a Microevolution Time Range in the Beet Armyworm

Abstract

In the case of the pests inhabiting metal polluted or fields where the use of pesticides is common, a natural selection of resistant individuals can occur. This may pose serious problems for humans, agriculture, as well as the economies of many countries. In this study, the hypothesis that multigenerational (120 generations) exposure to cadmium of a beet armyworm population could be a selecting factor toward a more efficient DNA protection was verified. The hemocytes of individuals from two culture strains (control and Cd-exposed) were treated with H2O2 (a DNA-damaging agent) or PBS (reference). The level of DNA damage was assessed using the Comet assay immediately and 5, 15 and 30 min. after the treatment. The immediate result of the contact with H2O2 was that the level of DNA damage in the hemocytes of the insects from both strains increased significantly. However, in the cells of the Cd-exposed individuals, the level of DNA damage decreased over time, while in the cells from the control insects it remained at the same level with no evidence of repair. These results suggest that efficient defense mechanisms may exist in the cells of insects that have prolonged contact with cadmium. Some evolutionary and trade-off aspects of the phenomenon are discussed. In a wider context, comparing the results obtained in the laboratory with field studies may be beneficial for understanding basic mechanisms of the resistance of an organism. To summarize, the high potential for the repair of DNA damage that was observed in the insects from the cadmium strain may confirm the hypothesis that multigenerational exposure to that metal may possibly contribute to the selection of insects that have a wider tolerance to oxidative stress. However, our investigations of polymorphism using AFLP did not reveal differences between the two main insect strains.

Fig 1. Apoptosis profile in exemplary hemolymph samples of the 5^th instar of Spodoptera exiqua from the control and cadmium strains as measured by the Muse^™ Annexin V and Dead Cell assay.

Abbreviations: Variant 0 –individuals from the control and cadmium-treated strains through 120 generations; Variant 1 –individuals from the control and cadmium strains that were fed the exchanged diet (control to cadmium or cadmium to control) for one week. Variant 2 –individuals from the control and cadmium strains that were fed the exchanged diet for one generation. The lower left (LL) quadrant represents normal healthy cells; the lower right (LR) and upper right (UR) quadrants represent early and late apoptotic cells, respectively; the upper left (UL) quadrant represents necrotic cells.

Fig 2. Apoptosis profile (a)–%; mean of 12 or 6 measurements in variant 0 or variants 1 and 2, respectively and total apoptotic cells (b)–mean ± SD in the hemolymph samples of the 5^th instar of Spodoptera exiqua from the control and cadmium strains.

Abbreviations: Variant 0 –individuals from the control (C) and cadmium-treated (Cd) strains through 120 generations; Variant 1 –individuals from the control and cadmium strains that were fed the exchanged diet (control to cadmium (C->Cd) or cadmium to control (Cd->C)) for one week. Variant 2 –individuals from the control and cadmium strains that were fed the exchanged diet for one generation. Stars denote statistically significant differences between the C->Cd group in variant 2 and the C->Cd group in variant 1 or the Cd group in variant 0 (ANOVA, Tukey test, p < 0.05).

Fig 3. Mean ± SD concentration of hydrogen peroxide in the hemolymph samples of the 5^th instar of Spodoptera exiqua from the control and cadmium strains.

Abbreviations: Reference—concentration of H₂O₂ in the samples without hemolymph, Variant 0 –individuals from the control (C) and cadmium-treated (Cd) strains through 120 generations; Variant 2 –individuals from the control and cadmium strains that were fed the exchanged diet for one generation. The same letters indicate homogenous strain-groups (ANOVA, Tukey test, p < 0.05).

Fig 4. Tail DNA (%; mean ± SD) in the nuclei of the hemocytes of the 5^th instar of S. exigua from the control and cadmium strains (Variant 0). After isolation, the cells were suspended in PBS and mixed with a H₂O₂ solution (treated groups; final concentration 50 μM) or with PBS (reference groups) and incubated for 1 min.

Abbreviations: ○ or ■ –mean of the medians of fifty nuclei that were measured on each slide; 0, 5, 15 or 30 min—time period after the end of the incubation; the same letters indicate homogenous groups within a strain (ANOVA, Tukey test, p < 0.05).

Fig 5. DNA comet images in the hemocytes of the 5^th instar of S. exigua from the control and cadmium strains. After isolation the cells were suspended in PBS and mixed with a H₂O₂ solution (treated groups; final concentration 50 μM) or with PBS (reference groups) and incubated for 1 min.

Abbreviations: Variant 0 –individuals from the control and cadmium-treated strains through 120 generations; Variant 1 –individuals from the control and cadmium strains that were fed the exchanged diet (C->Cd: control to cadmium or Cd->C: cadmium to control) for one week. Variant 2 –individuals from the control and cadmium strains that were fed the exchanged diet for one generation; Time—time period (0, 5, 15 or 30 min) after the end of the incubation with H₂O₂ or PBS.

Fig 6. Tail DNA (%; mean ± SD) in the nuclei of the hemocytes of the 5^th instar of S. exigua from the control and cadmium strains that were fed the exchanged diet (control to cadmium or cadmium to control) for one week (Variant 1). After isolation the cells were suspended in PBS and mixed with a H₂O₂ solution (treated groups; final concentration 50 μM) or with PBS (reference groups) and incubated for 1 min.

Abbreviations: ○ or ■ –mean of medians of fifty nuclei measured on each slide; (C->Cd), (Cd->C)–individuals from the control and cadmium strains that were fed the exchanged diet (control to cadmium or cadmium to control, respectively); 0, 5, 15 or 30 min—time period after the end of the incubation; the same letters indicate homogenous groups within a strain (ANOVA, Tukey test, p < 0.05).

Fig 7. Tail DNA (%; mean ± SD) in the nuclei of the hemocytes of the 5^th instar of S. exigua from the control and cadmium strains that were fed the exchanged diet (control to cadmium or cadmium to control) for one generation (Variant 2). After isolation the cells were suspended in PBS and mixed with a H₂O₂ solution (treated groups; final concentration 50 μM) or with PBS (reference groups) and incubated for 1 min.

Abbreviations: ○ or ■ –mean of medians of fifty nuclei measured on each slide; (C->Cd), (Cd->C)–individuals from the control and cadmium strains that were fed the exchanged diet (control to cadmium or cadmium to control, respectively); 0, 5, 15 or 30 min—time period after the end of the incubation; the same letters indicate homogenous groups within a strain (ANOVA, Tukey test, p < 0.05).