Study On The Detoxification Mechanism And Oxidative Stress Of Eisenia Fetida Under Cadmium Stress

Earthworms are more sensitive to most pollutants than other soil animals.It can be used as early warning organisms to evaluate soil pollution and soil environmental quality.The high concealment,irreversibility and long-term accumulation of heavy metals lead to the varying destruction of soil ecosystems.In this study,Eisenia fetida was used as the test animal.The toxic symptoms,behavior and growth inhibition rate of earthworms under cadmium(Cd)stress were observed,the indices of oxidative stress in earthworms were determined,and the effects of oxidative stress response after different stress times and concentrations were analyzed.At the same time,the effects of amino acid metabolism in the head and tail tissues of earthworms and the changes of carbon utilization of microbial communities in earthworm and soil after Cd stress were studied.Then,the driving mechanism of earthworm to its physiological functions was explored by combining the effects of oxidative stress,amino acid metabolism and carbon utilization.With the help of macro gene sequencing,the regulation mechanism between microbial communities in earthworm and in soil was analyzed.Thus,the detoxification mechanism of earthworm under Cd stress was revealed.This provides scientific basis for the ecological safety risk assessment of Cd contaminated soil and the development and application of bioremediation technology.The results are as follows:(1)Cd had a certain toxic effect on earthworms,and they were sensitive to Cd stress,the toxic reaction showed a significant dose-time relationship.With the increase of Cd concentrations,the degree of toxicity increased,but decreased slightly locally.When the toxicity of Cd exceeded that of earthworms,worms were died.(2)In the short-term group of artificial soil test,for the tail tissue of earthworm,the main oxidative stress indices that could be used for early warning were CAT,TP,SOD and POD.For the head tissue,they were TP,CAT and POD;in the long-term group,the main indices of tail tissue were GPX and MDA,while GPX in the head tissue.In the natural soil test,for the tail tissue,the indices that sensitively and effectively indicate the toxicity of Cd stress were CAT,TP and MDA,while POD in the head tissues of earthworms.(3)The improved factor analysis,combination of principal component analysis and traditional biostatistics and the combination of analytic hierarchy process and factor analysis could establish the screening models of biomarkers,which could be used to screen oxidative stress indices for establishing a rapid,low-cost,accurate and scientific ecology safety assessment of heavy metal pollution.This made an accurate and scientific evaluation of ecological pollution and biomarkers screening.(4)Cd stress altered the metabolic pathway of earthworms and the amino acid composition.At the same time,Cd stress also changed the functional diversity of the microbial communities in the soil and in the earthworms.With an extension of stress time and an increase in stress concentration,earthworms will adjust their own physiological functions(including the microbial community structure and stress mechanisms in the body)and regulate the microbial community structure in the external environment to obtain the necessary substances for growth.Aspartic,glutamic,cystine,methionine,histidine in earthworm head tissue and the cystine,phenylalanine in earthworm tail tissue were important amino acids for maintaining the earthworm physiological metabolism and resisting Cd stress.2-hydroxybenzoic acid,?-hydroxybutyric acid,glutamyl-L-glutamic acid,?-butyric acid,threonine and ?-cyclodextrin were important carbon sources for the earthworm to maintain their normal physiological metabolism after Cd stress.(5)Making the path analysis for the changes of physiological function in earthworm via stepwise regression method,which revealed the driving process in earthworm to its oxidative stress effects and functional diversity of microbial communities under Cd stress: Under 50 mg/kg Cd stress,the ACh E in earthworm could regulate non-dominant populations with uniform distribution in microbial communities.At 100-125 mg/kg,MDA was closely related to the dominant population,which mainly used D-galacturonic acid as carbon source.At 250 mg/kg,the microbial communities in earthworm mainly used glycyl-L-glutamic acid to obtain glutamic acid,which was supplied to the organs to synthesize GSH.At the highest concentrations(500 mg/kg),oxidative stress in earthworm was mainly used to maintain its basic physiological metabolism;the original microbial communities were completely degraded,the pioneer species which could tolerant to Cd were appeared.And the pioneer species was closely related to GPX and CAT,its main carbon source was glycyl-L-glutamic acid.At the same time,it was found that oxidative stress was more sensitive and effective than the microbial communities during short-term stress,and the former regulated the latter.In the long-term group,the functional diversity of microbial communities may be regulated by other factors.(6)This study integrated the canonical correlation ideas into TOPSIS method to establish a new mathematical analysis model.The model used the changes of carbon utilization intensity as raw data to analyze the characteristics of microbial communities under different stress times and concentrations,determine the regulatory time node and concentration node,and explore the regulation process of earthworms on microbes in vivo and soil under Cd stress,and investigate the succession of microbes with stress time.Results found the regulation process of the microbial communities between the earthworms and soil under Cd stress can be divided into the following five stages: During the first stage(1-3 days),the microbial community in the earthworms regulated the soil microbial community,but on the third day,the regulation was weakened.During the second stage(day 4),the soil microbial community was not only affected by the microbes in earthworms but also by the increasing Cd stress intensity.During the third stage(day 5),the microbial communities in earthworms and soil were both greatly affected by Cd poisoning,and the microbes were in a transition stage from stable to declining.During the fourth stage(6-7 days),the microbes in earthworms were able to regain control of the soil microbes once again,and the Cd-tolerant microbes began to appear and proliferate.During the fifth stage(8-10 days),the regulation of soil microbes by microbes in earthworms weakened,while the Cd-tolerant population in soil microbial communities gradually evolved in this stage to adapt to the increasing Cd stress.During the long-term stress tests,the difference between the microbial communities in soil and in earthworms increased,and there was almost no regulation between them,which may form two communities with different functions.(7)With the help of the second generation macro gene sequencing technology,the functional genes of microbes in earthworms and in soil under short-term Cd stress of 100 mg/kg were analyzed.The results showed that HBA gene,NEUROD1 gene and ABCA3 gene were the regulatory genes of microbial community in earthworms under Cd stress,while TC.FEV.OM gene and che BR gene were the main regulatory gene of microbial community in soil.At the same time,the Cd-tolerant microbes in the fifth regulatory stage were the new unnamed species in the Massilia.