Comparative Study On The Growth Inhibition Effect And Mechanism Of Raphidocelis Subcapitata Under The Stress Of Roxithromycin And Lincomycin

Due to the wide application of antibiotics and their frequent detection in the aquatic environment,the potential impact of antibiotics on the environment has become an important topic for discussion and research.Antibiotics discharged into water environment will directly affect sensitive primary producers-algae,and they are usually used as indicator organisms for water quality indicators and chemical assessment.Therefore,studying the effect and mechanism of antibiotics on algae is helpful to understand the negative effects of antibiotics on the whole ecosystem more scientifically.In recent years,Roxithromycin（ROX）and lincomycin（LIN）have been frequently detected in water environment,and their concentrations in surface water even reach microgram level,which means that the risks to aquatic organisms should not be underestimated.Because ROX and LIN have the same mechanism of action and antibacterial activity to bacteria,some scholars speculated that they have the same target for algae.However,it was found that the sensitivity of algae to two antibiotics is quite different,there are different EC₅₀ values of these two antibiotics to algae.Therefore,this paper takes ROX and LIN as research objects,and try to explore the inhibitory effects of two antibiotics on the growth of Raphidocelis subcapitata（R.subcapitata）,and using the second generation sequencing technology to reveal the mechanism of action of two antibiotics on R.subcapitata at the molecular level,and verifing the transcriptome results from the physiological characteristics such as oxidative stress and photosynthesis.Finally,the mechanism of action of the two antibiotics on algae was compared from the molecular level and physiological level,and the reason of the difference in sensitivity of the two antibiotics was analyzed.The research results are as follows:（1）The results of growth inhibition experiment showed that the EC₅₀value of ROX was0.05 mg/L,which was much lower than 0.29 mg/L of LIN after exposure to two antibiotics for96 hours,and R.subcapitata was more sensitive than LIN.（2）Transcriptome analysis of ROX suggested that ROX would not pose a threat to green algae at the environmental exposure level.Under the high concentration of ROX（0.09 mg/L）,2306 differential genes（983 up-regulated and 1323 down-regulated）were detected,which were mainly enriched in energy,lipid,vitamin and DNA replication and repair.However,there were no changes in the pathways related to translation,protein translocation and processing.Unexpectedly,we found that the genes involved in the repair process of base excision were inhibited,which indicated that ROX might be genotoxic and lead to DNA damage of R.subcapitata.The differences indicate that the mode of action of ROX in bacteria may not be inferred to green algae.（3）Transcriptome analysis of LIN suggested the pathways of photosynthetic antenna protein and porphyrin and chlorophyll metabolism pathways were promoted in R.subcapitata in environmental concentrations（0.0005 mg/L）of LIN treatment groups,while there were no change in algae growth.However,in the high-LIN treatment groups,it was observed that the DEGs related to carbon fixation pathway were up-regulated in photosynthesis pathway.In addition,the enriched pathways including nucleotide metabolism（purine and pyrimidine metabolism）,amino acid metabolism and carbohydrate metabolism were detected in the high LIN treatment group.Furthermore,DEGs changes involved in antioxidant defense process indicated that LIN caused oxidative stress in algae.Transcriptome analysis showed that LIN had different modes of action between R.subcapitata and bacteria.（4）Biomarkers related to photosynthesis and oxidative stress were determined by biological kit and microplate reader.The results showed that malondialdehyde（MDA）,superoxide dismutase（SOD）increased significantly and catalase（CAT）decreased significantly in the high concentration treatment groups of the two antibiotics,which indicated that the antioxidant mechanism of R.subcapitata was in common caused by these two antibiotics.In addition,it was found that the chlorophyll content of R.subcapitata showed an opposite trend with the change of concentrations under these two antibiotics,which showed that the photosynthesis of algae was affected.The results of physiological verification showed that both antibiotics can inhibit algae photosynthesis and induce oxidative stress in algae cells,which in accordance with the results of transcriptome analysis.（5）The mechanism of action of two antibiotics on R.subcapitata was compared at molecular level and physiological level.The results showed that the same pathways enriched in both treatment groups,which consist of membrane synthesis,antioxidant system,carbon fixation,amino acid metabolism and nucleotide metabolism.In addition,ROX also led to the enrichment pathways involved of photosynthesis,lipid metabolism,chlorophyll and cofactor metabolism,steroid and ketone synthesis,DNA replication and repair.This result indicated that ROX had more targets than LIN on R.subcapitata.The results of physiological comparison showed that ROX caused more severe changes and higher peak values of various enzymes,which indicated that the oxidative stress caused by ROX was stronger.The comparison between the above two levels can explain the algae is more sensitive to ROX,and its EC₅₀ is lower than LIN.