Generation Genotoxic Effects Of Systemic Pesticides On Mitochondria Of Earthworm

Systemic pesticides are a kind of broad-spectrum pesticides with internal absorption and conductivity.It can be distributed in plants through penetration and conduction,and effectively control pests and bacteria feeding on crops.It is widely used in global agricultural production.However,many kinds of systemic pesticides will remain in environmental media,which may have toxic effects on non-target organisms,resulting in a series of ecoenvironmental health and safety problems.Previous studies in our laboratory found that systemic pesticides will have generation genotoxic effects on bee mitochondrial DNA(mtDNA),that means the toxic effects can be transmitted to offspring through the genetic effect of mtDNA,and finally develop into malignant ecological events.However,there is still a lack of systematic research on the generation toxicity of pesticides,especially the potential toxicity of other non-target organisms has not been reported.Earthworms are an important part of soil ecosystem that play a vital role in improving soil fertility and promoting crop growth.They are very sensitive to pollutants in soil and is widely used in biological toxicology research.Previous studies have shown that systemic pesticides will have obvious physiological toxicity to earthworms,so the purpose of this study was to study whether systemic pesticides would have a significant negative impact on the mtDNA of earthworms and transmit it on to offspring earthworms.In this study,Eisenia foetida was used as the model organism,and four neonicotine insecticide(clothianidin,thiamethoxam,thiacloprid and acetamiprid),two systemic fungicides(difenoconazole and azoxystrobin)and eight mixed pesticides combined with them were selected as the exposure pollution sources of mother earthworms.Two generations of earthworms(F0,F1)were cultured in natural soil.The generation genotoxic effects of systemic pesticides on earthworm mitochondria were revealed by comparing the changes of mitochondrial DNA copy number(mtDNAcn)of the two generations of earthworms.Then we have evaluated the effect of systemic pesticides on the maximum mitochondrial respiratory rate(StateⅢu)of earthworms,analyzed the correlation between earthworm mtDNA damage and mitochondrial respiratory function,and explained the direct manifestation and consequences of mitochondrial DNA damage.Thus it provides a theoretical basis for the biological risk assessment and safe use of systematic pesticides.The main results are as follows:(1)The earthworms of F0 generation were exposed to the recommended dose of single and mixed pesticides for 30 days,and the mtDNAcn of earthworms in each group was measured.The results showed that 2 neonicotine insecticides,2 systemic fungicides and 8 mixed pesticides had obvious toxic effect to mtDNA of earthworms,which was manifested in the inhibition of earthworm mtDNAcn.In the neonicotine insecticide groups,the toxicity of thiamethoxam was significantly greater than that of clothianidin.And thiacloprid and acetamiprid had no significant toxicity to earthworm mtDNA.The negative toxic effects of difenoconazole and azoxystrobin were consistent,but the toxic effects were significantly lower than those of clothianidin and thiamethoxam.The eight mixed pesticides had inhibitory effects on earthworm mtDNAcn,and the toxic effects of different types of mixed pesticides were different.Among them,acetamiprid+azoxystrobin mixed pesticides had additive toxicity on earthworm mitochondrial DNA(p<0.05),and the other seven mixed pesticides had no significant additive toxicity,but the inhibitory effect on mtDNAcn was higher than that of single systematic pesticides.(2)The earthworms of F1 generation grew in pollution-free environment.The mitochondrial mtDNAcn of earthworms in each group was measured,and the changes were compared to mtDNAcn of earthworms in the two generations.The results showed that the effect of single systemic pesticides on mtDNA of F0 generation earthworm could be inherited stably,among which clothianidin and thiamethoxam had generation genotoxic effect on mtDNA of earthworm,and mtDNAcn of F1 generation earthworm also decreased relatively.The mtDNA of F1 earthworm treated with thiacloprid and acetamiprid has not been affected,which indicated that the two pesticides would not have toxic effect on earthworm DNA at the experimental dose.The mtDNAcn of F1 generation earthworms in difenoconazole and azoxystrobin groups were relatively reduced,which indicated that difenoconazole and azoxystrobin had certain generation genotoxicity to mtDNA of earthworms.Among the 8 groups treated mixed pesticide,the mtDNAcn of F1 generation earthworms in thiazoline+azoxystrobin group decreased significantly,and the mtDNAcn of F1 generation earthworms in the other 7 groups decreased to a certain extent,which indicated that mixed systemic pesticides have generation genotoxic effects on mtDNA of earthworms;However,the reduction of mtDNAcn in F1 generation was lower than that in F0 generation,which indicated that the toxic effect of mixed pesticides decreased relatively with intergenerational inheritance.(3)It is to explore the effects of systemic pesticides on mitochondrial respiratory function of two generations of earthworms.The results showed that for the single systemic pesticide group,the StateⅢu of F0 generation earthworm don’t show significant toxic effects,but fluctuated to a certain extent,indicating that the toxic effect of single systemic pesticide on the mitochondrial respiratory function of the mother generation earthworm was relatively weak.The StateⅢu of F1 generation earthworm in the single systemic pesticide group was inhibited,and the groups most affected by toxicity were thiamethoxam,thiamethoxam and azoxystrobin.The StateⅢu of two generations of earthworms in the mixed pesticide group was relatively reduced,which indicated that the mixed pesticide had an effect on the respiratory function of earthworm mitochondria and had the characteristics of epigenetics.In the treatment group and the control group,the StateⅢu of the F1 generation earthworm is higher than F0,which indicated that the respiratory function of the earthworm may recover with the change of the environment.But the growth of StateⅢu of the F1 generation earthworm in the control group is higher than that in the treatment group,which indicated that the recovery of mitochondrial respiratory function is relatively weaker due to pesticide exposure.In addition,correlation analysis showed that there was no correlation between mtDNA damage and mitochondrial respiratory function of two generations of earthworms in the single systemic pesticide group.But for the mixed pesticide group,the two biomarkers had a positive correlation in F0 generation and a negative correlation in F1 generation.The reasons for the inconsistency of this correlation need to be further studied.