Toxicity Study Of Microcystins-LR(MC-LR) On Planarian Dugesia Japonica

Due to global climate changes and increasing human activities,the content of phosphorus(P)and nitrogen(N)in water has increased sharply,and the eutrophication of the water has intensified,which has resulted in the rapid growth of cyanobacteria and serious pollution of streams and lakes.Microcystins(MCs)are one kind of the secondary metabolites of cyanobacteria,with multiple isomers,of which Microcystins-LR(MC-LR)is the most widely distributed and most toxic toxin.The planarian Dugesia japonica is widely distributed in the freshwater ecological environment of China.It has simple body structure and strong tissue regeneration ability,and it is extremely sensitive to aquatic ecosystem toxicants,so it can be used as a model animal for toxicological researches.At present,the toxicity studies of MC-LR mainly focus on mammals and fishes,the specific mechanism of MC-LR is not clear,and the toxicity of MC-LR on planarians has not been reported.In this paper,we use the planarian Dugesia japonica as an experimental material to explore the toxicity of MC-LR on intact worms and regenerative fragments,in order to provide reference for the toxicity of MC-LR to aquatic invertebrates and the risk of aquatic ecological environment of MC-LR.In this paper,the toxic effects of MC-LR on intact worms and regenerative fragments are explored by lethal concentration 50% measurement,biochemical level detection,q RT-PCR,immunofluorescence,bioinformatics,RNAi,WISH and so on.The results are as follows:1.The result of LC50 test showes that the 96 h LC50 of MC-LR on planarian Dugesia japonica is 3.557mg/L and the 95% confidence interval is 3.376 3.756 mg/L.2.The results of biochemical level test show that the activities of antioxidant enzymes SOD,CAT,GSH-Px,GSH-ST,MDA content and T-AOC level all change in different degrees after different concentrations of MC-LR exposing for different time.T-SOD activity increases significantly in the early and middle stages of MC-LR exposure,while CAT activity decreases at first and then increases during MC-LR exposure.After 0.2 mg/L and 0.4 mg/L MC-LR exposure,GSH-Px activity increases in the early stage of exposure,while GSH-Px activity and T-AOC level decrease in the later stage of MC-LR exposure.GSH-ST activity and MDA content increase on 3 d after 0.2 mg/L MC-LR exposure,and continue to increase during 0.4 mg/L MC-LR exposure.After 0.9 mg/L MC-LR exposure,GSH-ST activity decreases at first and then increases.In the middle and late stages of exposure,GSH-Px activity and MDA content show a decreasing-increasing-decreasing trend,and T-AOC level continues to decrease.The results show that MC-LR affects the antioxidation capacity of planarian and induces oxidative damage.3.The results of morphology show that after MC-LR exposure,head regenerative fragments are normal,intact worms and tail regenerative fragments appear prepharyngeal albinism,and finally disintegrate.4.The results of q RT-PCR and immunofluorescence show that MC-LR inhibites the expression of PP2 A,inhibites the proliferation and differentiation of neoblasts,and promotes apoptosis.Futhermore,MC-LR does not affect the development of nervous system on planarians.5.The results of RNAi and immunofluorescence show that DjTYR is not related to the pigment synthesis of planarian Dugesia japonica,and DjAlbino and DjPBGD can inhibit the synthesis of body pigments without affecting the proliferation of stem cells on D.japonica after knocking down.The results of q RT-PCR and WISH show that MC-LR affects the expression and distribution of DjAlbino and DjPBGD.The results indicate that MC-LR inhibites the proliferation and differentiation of neoblasts by inhibiting the expression of PP2 A,and gives rise to apoptosis by inducing oxidative damage and inhibiting the expression of PP2 A,resulting in the final disintegration of intact worms and tail regenerative fragments.In addition,MC-LR impacts the differentiation of neoblasts into pigment cells by affecting the expression and distribution of DjAlbino and DjPBGD,leading to albinism of intact worms and tail regenerative fragments.