Title Page

ABSTRACT

Contents

1. Introduction 10

2. Materials and Methods 15

2.1. Preparation of chemicals 15

2.2. Marine mysids culture 15

2.3. Acute toxicity test 15

2.4. RNA extraction and sequencing 16

2.5. De novo assembly and functional annotation 16

2.6. Expression profiling and differential gene expression analysis 17

2.7. Experimental validation by real-time quantitative reverse transcription PCR (qRT-PCR) 18

2.8. Protein extraction and measurement of oxidative parameters 18

2.9. Acridine orange (AO) and fast green (FG) staining 22

2.10. Statistical analysis 22

3. Results 26

3.1. Toxic effects of biocides in N. awatschensis 26

3.2. Transcriptome profiling of biocides in N. awatschensis 30

3.2.1. Differential gene expression of N. awatschensis exposed to biocides 30

3.2.2. Gene ontology (GO) enrichment analysis of N. awatschensis exposed to biocides 40

3.2.3. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of N. awatschensis exposed to biocides 45

3.2.4. Stress-related DEGs of N. awatschensis exposed to biocides 49

3.2.5. Validation of transcriptome profiles by qRT-PCR 53

3.3. Oxidative stress parameter measurement of N. awatschensis exposed to biocides 55

3.4. DNA damage measurement of N. awatschensis exposed to biocides 67

4. Discussion 70

5. Conclusion 75

References 76

국문초록 81

Table 1.1. Concentrations of diuron and cybutryne detected in the environment. 14

Table 2.1. Functional annotation summary 24

Table 2.2. Primers of overlapping genes for validation 25

Table 3.1. 96 h-LC₅₀ values from 96 h acute toxicity tests of diuron and cybutryne on aquatic organisms 29

Table 3.2. List of stress-related DEGs in N. awatschensis exposed to diuron and cybutryne 52

Fig. 1.1. Graphical abstract 13

Fig. 2.1. The chemical structures of diuron and cybutryne 23

Fig. 3.1. Survival rates of N. awatschensis exposed to diuron and cybutryne 27

Fig. 3.2. Concentration-response curves of N. awatschensis exposed to diuron and cybutryne A) is concentration-response curve of N. awatschensis at 96 h after exposure to diuron. B) is... 28

Fig. 3.3. Venn diagram of N. awatschensis exposed to diuron and cybutryne 31

Fig. 3.4. Venn diagram of N. awatschensis exposed to 100 μg/L and 1000 μg/L of diuron 33

Fig. 3.5. Networks of overlapping genes in 100 μg/L and 1000 μg/L of diuron 34

Fig. 3.6. Venn diagram of N. awatschensis exposed to 2 μg/L and 20 μg/L of cybutryne 36

Fig. 3.7. Networks of overlapping genes in 2 μg/L and 20 μg/L of cybutryne 37

Fig. 3.8. Volcano plots of N. awatschensis exposed to diuron and cybutryne 39

Fig. 3.9. GO enrichment analysis in N. awatschensis exposed to diuron 42

Fig. 3.10. GO enrichment analysis in N. awatschensis exposed to cybutryne 44

Fig. 3.11. KEGG pathway analysis in N. awatschensis exposed to diuron 46

Fig. 3.12. KEGG pathway analysis in N. awatschensis exposed to cybutryne 48

Fig. 3.13. Heatmap showing stress-related DEGs in N. awatschensis exposed to diuron and cybutryne 51

Fig. 3.14. Correlation of relative fold changes between qRT-RCR and RNA-seq results of N. awatschensis exposed to diuron and cybutryne 54

Fig. 3.15. Superoxide dismutase (SOD) and catalase (CAT) activity of N. awatschensis exposed to diuron. 56

Fig. 3.16. Glutathione peroxidase (GPx) and glutathione reductase (GR) activity of N. awatschensis exposed to diuron. 58

Fig. 3.17. Superoxide dismutase (SOD) and catalase (CAT) activity of N. awatschensis exposed to cybutryne. 60

Fig. 3.18. Glutathione peroxidase (GPx) and glutathione reductase (GR) activity of N. awatschensis exposed to cybutryne. 62

Fig. 3.19. Glutathione (GSH) and malondialdehyde (MDA) contents of N. awatschensis exposed to diuron. 64

Fig. 3.20. Glutathione (GSH) and malondialdehyde (MDA) contents of N. awatschensis exposed to cybutryne. 66

Fig. 3.21. AO and FG staining of N. awatschensis exposed to control, DMSO, and different concentrations of diuron and cybutryne for 24 h. Scale bar is 1000 μm, n＝3. 68

Fig. 3.22. Red/Green ratio in N. awatschensis exposed to control, DMSO, and different concentrations of diuron and cybutryne for 24 h. The standard error of the mean is shown by... 69