Study On The Toxicity Of Cyhalofop-Methyl To Different Developmental Stages Of Yellow River Carp And Its Combined Toxicity With 2-Methyl-4-Chlorophenoxy Acetic Acid

Fish farming in paddy fields are becoming more and more popular in modern agriculture,but pesticides sprayed in the paddy fields will remain in the water,which may adversely affect the farmed fish.In addition,pesticides are often applied to crops in the form of mixtures,so the combined toxic effects of multiple pesticides have been considered an important consideration in ecotoxicology.However,the current research mainly focuses on the effects of a single pesticide on the antioxidant system and inflammatory factors in the nucleus of fish,but little is known about the singled or combined toxicity of pesticides on the TLR signaling pathway,which is resists drug stress and transmits signals to activate the immune response in the cell’s nuclear.Yellow River carp,as China’s economic and precious freshwater fish,is a common food on people’s tables,and it is an ideal material for aquatic toxicology experiments.Therefore,this study used Yellow River carp as an experimental animal model to investigated the toxicity effects of the herbicide cyhalofop-butyl commonly used in rice fields on the Yellow River carp embryos developmental stage,the toxicity effects of cyhalofop-methyl and it combination with 2-methyl-4-chlorophenoxy acetic acid(MCPA-Na)on the antioxidant enzyme systems and TLR signaling pathways in the juvenile stage.1.The present study focused on an assessment of the impacts of cyhalofop-butyl(0、0.4、0.8、1.2 、 1.6 and 2 mg/L)on Yellow River carp embryos.It was found that cyhalofop-butyl inhibited the hatching of the embryos,and the hatching rate decreased with higher concentrations of the herbicide.The mortality rate was increased on exposure to cyhalofop-butyl,and was significantly higher in the 1.6 and 2mg/L treatment groups over 48 h.All of the embryos of the 2 mg/L treatment group died within the 48 h post-hatching stage.And the transcription of several related to apoptosis were also influenced by cyhalofop-butyl exposure.Further,cyhalofop-butyl exposure leading to a series of morphological changes(pericardial edema,tail deformation,and spine deformation)in embryos,which were consistent with significant modifications in the associated genes.These results provided a scientific basis for further studies into the effects of cyhalofop-butyl on aquatic organisms.2.In this study,we investigated the effects of different concentrations of cyhalofop-butyl(0,0.49 and 0.98 mg/L)on the subacute toxicity of Yellow River carp juveniles.The results showed that the cyhalofop-butyl increased ROS and MDA contents.And increased the activity of SOD,CAT and GSH-Px and the expression of genes related to the antioxidant system(SOD,CAT,GSH)under low concentrations exposured to eliminate ROS produced by exposure to cyhalofop-butyl,but at high concentrations exposured of cyhalofop-butyl,its activity and the transcription level of related genes were reduced,so that excessive accumulation of ROS caused oxidative stress and tissue damage.Simultaneously,exposured to cyhalofop-butyl increased the expression of TLR20 and activated the cascade reaction of the TLR signaling pathway to cope with external stresses.Therefore,the expression of ligands and key genes of in the TLR signaling pathway(TRAF6,TAK1,AP1 and P50)were all up-regulated during the initial exposured of cyhalofop-butyl.And the up-regulated genes related to TLR signaling pathway can promote the secretion of cytokines(IL-1β,INF-γ,TNF-α)in the immune response through the NF-k B signaling pathway and the MAPK pathway,resulted in an increase in inflammatory response in the Yellow River carp.In addition,down-regulation of immune-related genes(C3,LYZ)leads to a decline in non-specific immune defenses.3.In this study,we researched the combined toxicity of cyhalofop-butyl and MCPA-Na on Yellow River carp juveniles.In order to further verify the combined toxic effects of the two herbicides,we also analyzed the difference effects of cyhalofop-butyl and it combination with MCPA-Na(0.98mg/L+139.2 mg/L)on the expression of genes related to antioxidant enzyme systems and TLR signaling pathways in Yellow River carp juveniles.The results indicated that the combination of cyhalofop-butyl andMCPA-Na is synergistic.The larger changes of gene expression of SOD,CAT and GSH under the combined exposured of cyhalofop-butyl and MCPA-Na relative to the single exposured of cyhalofop-butyl.And the expression of CAT and GSH almost all showed a gradual downward trend in the combined exposured group,but it first increased and then decreased in the single exposured group.However,the expression changes of TLR20,TRAF6,TAK1,AP1,and P50 in the combined exposured group and the single exposured group were basically the same,but the expression of these genes in the combined exposure group was high than the single exposure group.In addition,the expression levels of IL-1β,IFN-γ,and TNF-α at almost all time points in the combined exposured group were almost higher than the single exposured group,but the m RNA expression level of C3 and LYZ in the combined exposured group were basically low relative to the single exposured group.In summary,exposure to cyhalofop-butyl inhibited the hatching of Yellow River carp embryos,increased its mortality and leads to its abnormal development.In addition,exposure to cyhalofop-butyl resulted in abnormal production of ROS,increased MDA content,changed in antioxidant enzyme activity,and m RNA levels of genes related to oxidative stress in Yellow River carp juveniles.At the same time,exposure to cyhalofop-butyl can also induced the expression of pro-inflammatory factors in the nucleus by regulating the expression of important genes related to the TLR pathway signaling pathway,thereby further regulating the immune response of Yellow River carp juveniles.Our study also found that the combined use of cyhalofop-butyl and MCPA-Na has a greater toxic effect on Yellow River carp juveniles.