Effect Mechanism Of Sodium Fluoride On Growth And Energy Metabolism Of Hyphantria Cunea

Fluoride is one of the global pollutants.With the development of modern industry,fluorine pollution seriously endangers the health of animals and plants.Hyphantria cunea is a worldwide quarantine pest,feeding on various agricultural and forestry crops has caused great harm to China’s agriculture and forestry sectors and resulted in significant economic losses to many provinces and cities in China.In this study,the effects of sodium fluoride on the growth and energy metabolism of H.cunea were studied.Firstly,the impact of the median lethal concentration of sodium fluoride on oxidative stress and immune response-related enzyme activities,as well as the gene expression,enzyme activity and substance content related to energy metabolism in the tissues（midgut and fat body）of the 6^th instar larvae of H.cunea were studied.Secondly,the differentially expressed transcriptomes of the sodium fluoride median lethal dose treatment group and control group were constructed,and the differential expression genes were compared and analyzed.Based on this,the ferritin genes（HcFerHCH and HcFerLCH）of H.cunea were screened,and the response mechanism of ferritin genes to sodium fluoride stress was analyzed.The main results are as follows:1.The semi-lethal concentration was calculated to be 186μg/m L through the toxicity test of different concentrations of sodium fluoride on the first day of the 4^th instar of H.cunea.The6^th instar larvae of H.cunea were treated with 186μg/m L of sodium fluoride,and the body size and weight were reduced.Further studies showed that oxidative stress and energy metabolism disorder occurred in the midgut and fat body of H.cunea larvae treated with sodium fluoride（186μg/m L）.The levels of reactive oxygen species and lipid peroxides in the midgut and fat body of H.cunea treated with sodium fluoride（186μg/m L）for 48 h and 72 h were significantly increased.Over time,the activity of antioxidant enzymes catalase and superoxide dismutase were gradually lower than those of the control group.Glutathione peroxidase activity was continuously inhibited.The decrease of nitric oxide synthase activity and nitric oxide content resulted in impaired immunity of H.cunea against sodium fluoride toxicity,causing oxidative stress and immune response.In addition,sodium fluoride also affected the energy metabolism of H.cunea larvae.Firstly,sodium fluoride inhibited the synthesis of fatty acids in the midgut and fat body by regulating the expression of fatty acid synthesis key gene acetyl Co A carboxylase gene（ACC）and fatty acid synthase gene（FAS）.With the significant decrease in ATP content,sodium fluoride promoted the decomposition of triglyceride by increasing lipase activity,reducing triglyceride and affecting lipid metabolism.Secondly,the metabolism of carbohydrate in H.cunea was impacted by median lethal concentration of sodium fluoride.Under sodium fluoride stress,the m RNA transcription levels of trehalose hydrolase and trehalose synthase and the related enzyme activities were continuously regulated to adapt to energy changes.In the midgut and fat body of H.cunea,the content of trehalose and glucose showed a downward trend.Sodium fluoride reduced the energy supply of H.cunea tissues,resulting in a decrease in the resistance of the insect body to external pressure.2.The transcriptome libraries of midgut and fat body of 6^th instar larvae of H.cunea treated with control（ddH₂O）and sodium fluoride（186μg/m L）were constructed respectively,and 58521Unigenes were obtained.KEGG pathway enrichment analysis of DEGs in the midgut and fat body showed that differential genes were significantly enriched in the ferroptosis pathway.The function of GO in distributing DEGs was found to be significantly enriched in iron ion binding function.The differential genes HcFerHCH and HcFerLCH related to iron metabolism were screened.Among them,sodium fluoride（186μg/m L）inhibited ferritin expression in the midgut and fat body of H.cunea.At 48 h,the expression levels of HcFerHCH in the fat body and midgut were 56.26%and 28.76%of the control group,respectively.At 72 h,they decreased to 56.19%and 11.12%of the control group,respectively.The expression of HcFerLCH in the fat body was31.74%～85.74%of the control group.The expression level in the midgut was only 14.74%～30.03%of the control group.3.In this study,two ferritin genes（HcFerHCH and HcFerLCH）were cloned.The open reading frame（ORF）length was 666～690 bp,encoding 221～229 amino acids,and both had a signal peptide and an iron response element（IRE）.IRE can be folded into a typical stem-loop secondary structure.The phylogenetic tree showed that the ferritin gene of H.cunea was conserved in Lepidoptera insects.RT-qPCR analyzed the transcription levels of HcFerHCH and HcFerLCH genes.The results showed that HcFerHCH and HcFerLCH genes were expressed in all developmental stages and tissues of H.cunea,and were highly expressed in male adults and hindgut.4.HcFerHCH and HcFerLCH gene-specific dsRNA were microinjected into H.cunea larvae by RNAi technology,and the expression levels of HcFerHCH and HcFerLCH at different time points were determined.The results revealed that the expression level of ferritin in H.cunea was significantly inhibited at 48 h,72 h and 96 h with inhibition rate of 26.18%～99.67%.The sensitivity of gene silencing of H.cunea to sodium fluoride was determined by bioassay.The results showed that silencing the target gene significantly increased the sensitivity of H.cunea to insecticides and the iron content of H.cunea larvae was significantly decreased.The ferrous and reactive oxygen species（ROS）content increased significantly,resulting in iron deficiency and oxidative stress in the body and a significant increase in mortality.