Title Page
Contents
Graphical Abstract 12
Abstract 13
Introduction 15
CHAPTER 1. The Role of Commensal Microbes on The Longevity Effect of Dietary Restriction in D. melanogaster 18
1.1. Abstract 18
1.2. Introduction 19
1.3. Results 22
1.3.1. Removal of commensal microbes reduces the longevity effect of DR 22
1.3.2. Commensal microbes increase the fecundity and body weight of D. melanogaster in poor diet 26
1.3.3. Acetobacter is related to the longevity effect of DR in D. melanogaster 28
1.3.4. Acetobacter mediates the longevity effect of DR via IIS modulation 36
1.3.5. Acetate derived from Acetobacter mediates the longevity effect of DR 41
1.4. Discussion 48
CHAPTER 2. The Role of Commensal Microbes in the Anti-aging Effects of Dietary Restriction on Intestinal Stem Cells in D. melanogaster 59
2.1. Abstract 59
2.2. Introduction 60
2.3. Results 64
2.3.1. DR suppresses age-related ISC proliferation 64
2.3.2. Removal of microbes suppresses age-related ISC proliferation 68
2.3.3. DR does not suppress age-related ISC proliferation in axenic fly 70
2.3.4. Lactobacillus is related to DR-related ISC proliferation 72
2.3.5. DR-related ISC proliferation is mediated by TOR signaling 75
2.3.6. Lactobacillus mediates DR-related ISC proliferation via TOR signaling regulation 78
2.4. Discussion 82
CHAPTER 3. The Role of Commensal Microbes on the Longevity Effects of Aronia berry (Aronia melanocarpa) in D. melanogaster 86
3.1. Abstract 86
3.2. Introduction 87
3.3. Results 90
3.3.1. Supplementation of Aronia berry extends the lifespan of fruit fly 90
3.3.2. The antioxidant gene SOD1 mediates the longevity effect of Aronia berry 96
3.3.3. Removal of commensal microbes reduces the longevity and antioxidant effects of Aronia berry supplementation 98
3.3.4. Supplementation of Aronia berry alters the microbial flora of fly 104
3.3.5. Supplementation of Aronia berry induces an immune response in fly 112
3.3.6. The Toll pathway mediates the microbial alteration and longevity effect of Aronia berry in fly 121
3.4. Discussion 125
Discussion 134
Conclusion 136
CHAPTER 4. Materials and Methods 137
4.1. Fly husbandry 137
4.1.1. Drosophila stocks 137
4.1.2. Dietary supplements 137
4.1.3. Generation of axenic and gnotobiotic fly 138
4.2. Bacterial analysis 139
4.2.1. Bacterial stocks and culture 139
4.2.2. Bacterial growth 140
4.2.3. Colony forming unit analysis 140
4.2.4. 16S rRNA analysis 140
4.2.5. Illumina-sequencing analysis 141
4.3. Drosophila Physiology 141
4.3.1. Survivorship 141
4.3.2. Fecundity 142
4.3.3. Body weight 143
4.3.4. Feeding rate 143
4.3.5. Developmental viability 144
4.4. RNA analysis 144
4.4.1. Quantitative reverse transcription-PCR (qRT-PCR) 144
4.4.2. Library preparation and RNA-seq data processing 145
4.4.3. RNA-seq data analysis 145
4.5. Protein expression and activity 146
4.5.1. Immunoblotting 146
4.5.2. Immunohistochemistry 146
4.5.3. Enzyme activity 148
4.6. NMR spectroscopy analysis 148
4.7. ROS detection 149
4.8. Statistical analysis 149
References 152
Table 1.1. The lifespan and log-lank test results comparing yeast concentration under conventional or axenic conditions 52
Table 1.2. The lifespan and log-rank test results comparing conventional and axenic flies at the same yeast concentration 53
Table 1.3. The lifespan and log-rank test results of flies fed homogenate of conventional or axenic fly 54
Table 1.4. The lifespan and log-rank test results of flies inoculated with 10⁸ CFUs microbes under various concentration of yeast 55
Table 1.5. The lifespan and log-rank test results of flies inoculated with microbes by same O.D. under various concentration of yeast diet 56
Table 1.6. The lifespan and log-rank test results of InR and chico mutant flies under high yeast diet 57
Table 1.7. The lifespan and log-rank test results of flies inoculated with 10⁸ CFUs A.pomorum under various concentration of yeast 58
Table 3.1. The lifespan and log-rank test results of flies under various concentration of Aronia berry supplementation 127
Table 3.2. The lifespan and log-rank test results of SOD1-expressing flies supplemented with Aronia berry 128
Table 3.3. The lifespan and log-rank test results of flies supplemented with Aronia berry under conventional or axenic condition 129
Table 3.4. The survival time and log-rank test results of conventional or axenic flies supplemented with Aronia berry under oxidative stress condition 130
Table 3.5. Alpha diversity analysis of microbiota of flies supplemented with Aronia berry 131
Table 3.6. The log-rank test results of septic injury survival of conventional or axenic flies supplemented with Aronia berry 132
Table 3.7. The lifespan and log-rank test results of Dorsal-expressing flies supplemented with Aronia berry 133
Table 4.1. Composition of fly husbandry food 150
Table 4.2. Sequences of primers for PCR 151
Figure 1.1. Removal of commensal microbes reduces the longevity effect of DR 24
Figure 1.2. The longevity effect of DR is attributed to the presence of microbes 25
Figure 1.3. Commensal microbes increase the fecundity and body weight in poor diet 27
Figure 1.4. The microbial load altered as the yeast concentration changed 29
Figure 1.5. CFUs of flies after inoculated with microbes 31
Figure 1.6. Acetobacter is required for the lifespan extension response to DR (inoculated with 10⁸ CFUs) 33
Figure 1.7. Acetobacter is required for the lifespan extension response to DR (inoculated with the same O.D. concentration) 35
Figure 1.8. Commensal microbe is associated with the modulation of IIS by DR 38
Figure 1.9. Re-association of Acetobacter increases the IIS in axenic fly 39
Figure 1.10. Acetate decreased by DR in conventional fly but not in axenic fly 42
Figure 1.11. Supplementation of acetate restores IIS reduction in axenic fly 43
Figure 1.12. Microbial 16S rRNA gene of flies after inoculated with A. pomorum 45
Figure 1.13. Inoculating P3G5 did not rescue the survival of flies under a restricted diet 47
Figure 2.1. Intestinal stem cell in Drosophila gut 63
Figure 2.2. DR suppresses the age-related ISC proliferation in D. melanogaster 66
Figure 2.3. Removal of microbes suppresses the age-related ISC proliferation 69
Figure 2.4. Age-related ISC proliferation is not decreased by DR in axenic fly 71
Figure 2.5. Commensal microbes mediate the regulating age-related ISC proliferation by DR 74
Figure 2.6. TOR-mediated regulation of cell differentiation in the ISC lineage 76
Figure 2.7. DR-related ISC proliferation is mediated by TOR signaling regulation 77
Figure 2.8. Lactobacillus regulates p4EBP expression in ISCs of aged fly in response to nutritional changes 79
Figure 2.9. ISCs phenotype in Lactobacillus-inoculated flies resembles TOR mutant flies 81
Figure 3.1. Supplementation of Aronia berry extends lifespan of D. melanogaster 92
Figure 3.2. Supplementation of Aronia berry does not induce adverse effects on fly 94
Figure 3.3. The antioxidant gene SOD1 mediates the longevity effect of Aronia berry 97
Figure 3.4. Supplementation of Aronia berry did not extend the lifespan of axenic fly 99
Figure 3.5. Supplementation of Aronia berry failed to increase oxidative stress resistance in axenic fly 100
Figure 3.6. Removal of commensal microbes reduces antioxidant effects of Aronia berry supplementation 102
Figure 3.7. Supplementation of Aronia berry reduces the microbial load of fly 107
Figure 3.8. Supplementation of Aronia berry did not induce significant change in the microbial composition of fly 108
Figure 3.9. Aronia berry did not reduce in vitro bacterial growth 111
Figure 3.10. Supplementation of Aronia berry induces the immune response in fly 114
Figure 3.11. Supplementation of Aronia berry increases expression of AMP genes associated with Toll signaling pathway 118
Figure 3.12. Supplementation of Aronia berry increases survival against septic injury caused by S. aureus but not E. coli 119
Figure 3.13. Supplementation of Aronia berry reduces microbial load through modulation of Toll pathway 123
Figure 3.14. Toll pathway mediates the life extension by Aronia berry supplementation 124