Using Dugesia Japonica To Construct A Biological Monitoring Model For Chloroaniline Water Pollution

Background: Whole effluent toxicity approach(WET approach)is one of the standard methods for water quality monitoring,and the standardization of indicator organisms is the key to the application of the WET approach.Dugesia japonica,as an experimental animal for ecotoxicological research,is gradually being recommended as an indicator organism for the WET approach,but the lack of monitoring indicators hinders its promotion and application as an indicator organism.At the same time,the existing monitoring indicators can only qualitatively or manually quantify the individual behavior of the planarian,which greatly weakens the application convenience of the planarian as an indicator organism.Therefore,this study takes the toxicological influence of two chloroaniline water environmental pollutants to Dugesia japonica as the breakthrough point.Based on the research results of the toxicology of planarians at home and abroad,explore and quantify planarians as indicator organisms at the individual level,organ level and molecular level.This enriches the basic data of planarian toxicology research and provides a quantitative method for planarian water quality WET approach.Methods: In this study,Dugesia japonica was used as experimental animals,and 3,4-dichloroaniline(3,4-DCA)and p-chloroaniline(PCA)were used as toxicity factors.Based on the LC50 of the acute toxicity test,we studied the acute toxicity influence of 3,4-DCA and PCA to Dugesia japonica,the planarian locomotor velocity(p LMV),behavioral analysis,regeneration ability and RNA-sequencing?Results:(1)The 24 h and 96 h LC50(95CI%)of 3,4-DCA and PCA against planarians were 21.67(19.72-23.80)mg/L and 14.40(12.87-15.83)mg/L,8.64(7.89-9.30)mg/L and 4.06(3.66-4.48)mg/L.Acute toxic morphological changes include scrunching?skin hollow?pharynx protrusion and body disintegration?Acute toxic behavioral changes include corkscrew?c-shape?tentative left and right?headswing and squirming.(2)The result of the control group in the classic p LMV method was 136±13.8.High,medium and low concentrations of 3,4-DCA would cause the reduction of p LMV,and the difference was significant;only high concentrations of PCA would cause the reduction of p LMV.(3)The dependent variable analysis provided by behavioral analysis can not only be compared with the classic p LMV,but also can be used to explain the behavior of planarians and quantify the five abnormal behaviors of planarians under exposure to chloroaniline poisons.Behavioral analysis of Velocity dependent variables provides a new method for calculating p LMV.Our new method is consistent with the results of the classic p LMV method,which verifies the feasibility of behavioral analysis.The 5-minute Distance moved of the control group was 7779±219.6mm,which was significantly different from 3,4-DCA at high,middle and low concentrations,and only significantly different from PCA at high concentrations.The dependent variable of the Movement showed that after 24 hours of stimulation with medium and high concentration of 3,4-DCA and high concentration of PCA,the Movement of planarian was significantly different from that of the control group.The results of the dependent variable of the Turn angle showed that the high,medium and low concentrations of 3,4-DCA would increase the number of Turn angle of the planarian(P<0.05),and the higher the toxicant concentration,the stronger the correlation.However,only a high concentration of PCA will cause an increase in the number of Turn angle of the planarian.The Turn angle is used to quantify the abnormal behavior patterns that appear in the planarian acute toxicity test.We first tried to introduce the Rotation dependent variable into the planarian behavior study,and found that only the high concentration group of 3,4-DCA would cause a significant difference in the number of planarian rotations.(4)The high concentration of chloroaniline delays the regeneration of planarian eye spots and increases the time for planarian eye spot regeneration.(5)The RNA-seq results showed that 3,4-DCA and the control group co-expressed 22,793 genes,of which 14 were up-regulated genes and 5 were down-regulated genes.The GO classification analysis of differential genes shows that the most differential genes are related to the binding of molecular functions.The GO enrichment analysis showed that the more differential genes were enriched and the Q value was smaller were GTPase binding,proline dehydrogenase activity,proline catabolic process,proline metabolic process and so on.PCA and the control group co-expressed 22,798 genes,of which 16 were up-regulated genes and 8 were down-regulated genes.The results of GO classification analysis of differential genes showed that 9 genes are related to the cellular anatomical entity of cell components.The higher gene enrichment also has the catalytic activity of molecular function.GO enrichment analysis showed that the most enriched function of differential genes was cysteine-type peptidase activity.Among them,Djap₉3854 is a gene that regulates the locomotion of planarians,and it is also a gene that is up-regulated by 3,4-DCA and PCA.Conclusion: 1.Planarians are more sensitive indicator organisms than zebrafish.Behavioral analysis provides a new method for calculating p LMV,which may replace the traditional grid line counting method.2.With the different environmental poisons can cause similar behavioral changes in planarians and the behavior of the planarian appears before death,planarian abnormal behavior patterns can be quantitatively analyzed by the Turn angle dependent variable.3.For the first time,the Rotation dependent variable was introduced into the planarian behavior analysis and found that Rotation can be used as a new indicator of asymmetry in the planarian brain.4.The delayed effect of chloroaniline on planarian eyepoint regeneration provides an organ-level monitoring index.Its quantification relies on manual observation and counting.Therefore,further research is still needed to promote the automation of eyepoint regeneration index numerical counting.5.Djap₉3854 is a gene that regulates the locomotion of planarians,and is also a gene up-regulated by 3,4-DCA and PCA.It may be a molecular level indicator for planarians to measure chloraniline water pollution.6.Planarian,as the indicator organism of the wastewater comprehensive toxicity method with chloroaniline poison as the main body,monitoring indicators include: LC50,Distance moved,Movement,Velocity,Turn angle,Ratation,eye point regeneration and Djap₉3854 gene.