The Research About The Effect Of Cu²⁺ On Antioxidant Enzyme And Cells Apoptosis Of Hermetia Illucens Larva

Objective:We hope to research about the influence of hemolymph antioxidant enzyme and the cells apoptosis of Hermetia illucens larva stressed in the dietary of Cu²⁺ long-term,investigate the toxicology influence of Hermetia illucens larva stressed by Cu²⁺,and provide theoretical data for the effect of the system about dealing with livestock feces harmlessly utilize Hermetia illucens larva stress in Cu²⁺ long-term.Methods:Through adding different concentration of Cu²⁺?0,75,150,300,600,1200 mg/kg?in the artificial diets to feed Hermetia illucens larva for 5 generations.We detected the accumulation and excretion of Cu²⁺ of hemolymph of 6 instars Hermetia illucens larva in the 1st,3rd and 5th generation by Plasma atomic emission spectrometer detection;detected the content of metallothionein?MT?and phenol oxidase?PO?by enzyme linked immunosorbent assay,detected the the activity of Catalase from micrococcus lysodeikticus?CAT?and Total antioxidant capacity?T-AOC?by visible spectrophotometry;observed apoptosis morphology change of 6 instars Hermetia illucens larva in the 1st,3rd and 5th generation Respectively by onfocal laser scanning microscopy;observed the ultrastructural changes of blood cells by Transmission electron microscope;detected the blood cells apoptosis rate by flow cytometry;Observed the DNA damage of blood cell by Comet assay.Results:?1?The accumulation of Cu²⁺ in hemolymph of Hermetia illucens larva is closely associated with the content of Cu²⁺ in the diets.The content of Cu²⁺ in hemolymph of Hermetia illucens larva is increased with increase of the Cu²⁺ concentration and the generation.When Cu²⁺ concentration is 1200 mg/kg,The content of Cu²⁺ in hemolymph and residuum reach the peak at 5th generation.?2?Antioxidant defense system of Hermetia illucens larva is activated in low concentration,suppressed in high concentration.When the concentration of Cu²⁺ is 75-300 mg/kg,the content of MT,PO and the activity of CAT,T-AOC are increased with the increase of concentration of Cu²⁺ and showed a trend of rising with generation;When the concentration of Cu²⁺ within the scope of 600-1200 mg/kg,the content of MT,PO and the activity of CAT,T-AOC are reduced with the increase of concentration of Cu²⁺ and showed a trend of decreasing with generation.?3?Hermetia illucens larva blood cells can be induced to apoptosis when stressed by Cu²⁺.The results of confocal laser scanning microscopy showed that,with the increase of Cu²⁺ concentration and the generation,the apoptotic nuclei have obvious hollow,solid shrinkage,collapse and concentrations,When the concentration of Cu²⁺ is 1200 mg/kg,the apoptotic cell nucleus of Hermetia illucens larva disintigrated with obvious plum blossom shaped pieces.TEM results show that,When the concentration of Cu²⁺ is 75-300 mg/kg,the ultrastructure of the blood cell manifests that the amount of mitochondria and endoplasmic reticulum in the cytoplasm significantly increased,When the concentration of Cu²⁺ is 600-1200 mg/kg,the amount of mitochondria and endoplasmic reticulum significantly reduced,lysosome increased,organelles such as golgi apparatus and the ribosome were not obvious,when the concentration of Cu²⁺ is 1200 mg/kg,the nucleus was obvious splitting;With the increase of concentration of Cu²⁺ and generation,the more obvious changes of apoptotic cell ultrastructures,at 5th generation,nuclear chromatin condensated,nucleolus disappeared,cytoplasm condensed,a large number of empty vesicles appeared,organelles deduced,cell membrane stretched out a large number of pseudopodia and invagination,rupture,some contents leakage from it;Apoptosis rate increased with the increase of concentration of Cu²⁺ and showed a trend of rising with generation,within the same generation,early apoptosis rate increased with the increase of concentration of Cu²⁺,the change of middle-late apoptosis rate was not significant;The same concentration of Cu²⁺ but different generations between the treatment group,early apoptosis rate and middle-late apoptosis rate increased with the increase of generation.?4?When the concentration of Cu²⁺ is 1200 mg/kg,the degree of DNA damage increased with the increase of generation,comet experiment in the tail phenomenon.Conclusion:?1?Cu²⁺ can accumulate in the hemolymph of Hermetia illucens larva,The content of Cu²⁺ is increased with increase of the Cu²⁺ concentration and the generation.;And Hermetia illucens larva have certain ability of excretion of Cu²⁺.?2? Antioxidant enzyme activity of Hermetia illucens larva can be enhanced when low concentration of Cu²⁺?75-300mg/kg?stressed,inhibited when high concentration of Cu²⁺?600-1200mg/kg?stressed,and antioxidant enzyme activity of low concentration of Cu²⁺ treatment group increased with the increase of generation,antioxidant enzyme activity of high concentration of Cu²⁺ treatment group decreased with increasing of generation.?3?The blood cells of Hermetia illucens larva can be induced to apoptosis when Cu²⁺ stressed,with the increase of concentration and generation,the morphology and structure of apoptosis cells have significant change,early cell apoptosis rate is increased.?4?Cu²⁺ stress on Hermetia illucens larva can lead to DNA damage,when the concentration is 1200 mg/kg,we can observe that the DNA of Hermetia illucens larva has obvious trailing phenomenon.