Title Page

Contents

ABSTRACT 15

Chapter 1. General Introduction 19

General Introduction 20

The biology and classification of the kelp Saccharina japonica 21

Biochemical composition of Saccharina japonica 26

Macroalgae as a host for epibionts 26

The influence of sessile epifauna on seaweed substarta 28

Bryozoans 32

Bryozoans that live on Saccharina japonica 34

Hydrozoans 35

Research objectives 37

REFERENCES 39

Chapter 2. Does bryozoan colonization alter the biochemical composition of Saccharina japonica affecting food safety and quality? 51

ABSTRACT 52

INTRODUCTION 53

MATERIALS AND METHODS 55

Algal and bryozoan materials 55

Viability assay 55

Proximate composition 56

Amino acid composition 59

Fatty acid composition 61

Metal composition 62

Acute toxicity 63

Statistical analysis 64

RESULTS AND DISCUSSION 65

REFERENCES 72

Chapter 3. Effects of hydrozoans colonization on the biochemical compositions of the brown seaweed Saccharina japonica 82

ABSTRACT 83

INTRODUCTION 84

MATERIALS AND METHODS 86

Algal and hydrozoan materials 86

Viability assay 86

Proximate composition 86

Amino acid composition 87

Fatty acid composition 87

Metal composition 87

Acute toxicity 88

Statistical analysis 88

RESULTS AND DISCUSSION 89

REFERENCES 96

Chapter 4. Induced changes in the proteomic profile of the phaeophyta Saccharina japonica upon colonization by the bryozoan Membranipora membranacea 105

ABSTRACT 106

INTRODUCTION 108

MATERIALS AND METHODS 110

RESULTS AND DISCUSSION 113

REFERENCES 122

Chapter 5. Influences of hydrozoan colonization on proteomic profiles of the phaeophyta Saccharina japonica 134

ABSTRACT 135

INTRODUCTION 137

MATERIALS AND METHODS 139

Seaweed, hydrozoan, and reagents 139

Protein electrophoresis 139

Quantitative analysis 141

Protein digestion and identification 141

RESULTS AND DISCUSSION 143

REFERENCES 151

Chapter 6. Earlier colonization marker proteins identified from the phaeophyta Saccharina japonica upon colonization of bryozoans 165

ABSTRACT 166

INTRODUCTION 168

MATERIALS AND METHODS 171

RESULTS AND DISCUSSION 172

REFERENCES 194

Chapter 7. Earlier colonization marker proteins identified from the phaeophyta Saccharina japonica upon colonization of hydrozoans 223

ABSTRACT 224

INTRODUCTION 227

MATERIALS AND METHODS 229

RESULTS AND DISCUSSION 230

REFERENCES 249

SUMMARY AND CONCLUSION 274

ABSTRACT (KOREAN) 280

Table 1-1. Classification of Saccharina japonica 22

Table 2-1. Proximate compositions of healthy blades, colonized blades, and bryozoans separated from Saccharina japonica 77

Table 2-2. Total and free amino acid contents of healthy tissues, bryozoan colonized tissues, and bryozoans separated from Saccharina japonica 78

Table 2-3. Fatty acid compositions of healthy tissues, bryozoans-colonized tissues, and bryozoans separated from Saccharina japonica 79

Table 2-4. Metal contents in healthy tissues, bryozoan-colonized tissues, and bryozoans separated from Saccharina japonica 80

Table 3-1. Proximate compositions of healthy blades, hydrozoan-colonized blades, and hydrozoans separated from S. japonica 100

Table 3-2. Total and free amino acid contents of healthy tissues, hydrozoan-colonized tissues, and hydrozoans separated from Saccharina japonica 101

Table 3-3. Fatty acid compositions of healthy tissues, hydrozoan-colonized tissues, and hydrozoans separated from S. japonica 102

Table 3-4. Metal compositions of healthy tissues, hydrozoan-colonized tissues, and hydrozoans separated from S. japonica 103

Table 4-1. Identified proteins in the bryozoans-colonized tissue and healthy tissue of the late harvested Saccharina japonica. 127

Table 5-1. Identified proteins in the hydrozoan-colonized tissue and healthy tissue of the late-harvested Saccharina japonica. 156

Table 6-1. Identified proteins in the healthy tissue 1-㎝ away from the lately bryozoan-colonized tissue and healthy tissue of the late harvested Saccharina japonica. 208

Table 6-2. Identified proteins in the lately bryozoan colonized tissue and healthy tissue of the late harvested Saccharina japonica. 212

Table 7-1. Identified proteins in the healthy tissue 1-㎝ away from the lately hydrozoan colonized tissue of the late harvested Saccharina japonica. 257

Table 7-2. Identified proteins in the lately hydrozoan colonized tissue of the late harvested Saccharina japonica. 261

Figure 1-1. Names of tissue parts of the kelp Saccharina japonica 23

Figure 1-2. The kelp life cycle 25

Figure 1-3. Animals commonly found on different kelp species. 31

Figure 1-4. Kelp blades colonized by the bryozoans 33

Figure 1-5. Kelp blades colonized by hydrozoan 36

Figure 2-1. Tissue viabilities of healthy and colonized blades of Saccharina japonica. 81

Figure 3-1. Viabilities of healthy and colonized blades of Saccharina japonica. 104

Figure 4-1. 2-DE profiles of the late harvested S. japonica. 130

Figure 4-2. A close view of the identified up-regulated proteins (indicated by arrows) altered by colonization by M. membranacea. 131

Figure 4-3. A close view of the identified down-regulated proteins(indicated by arrows) altered by colonization by M. membranacea. 132

Figure 4-4. A close view of the identified proteins found mostly in the tissue colonized by M. membranacea but not in the healthy tissue (single asterisk) and identified proteins found mostly in healthy tissue but not in colonized tissue (double asterisks). 133

Figure 5-1. Two-DE profiles of the late-harvested Saccharina japonica. 160

Figure 5-2. A close view of the identified upregulated proteins (indicated by arrows) found mostly in the hydrozoan-colonized tissue but rare in the healthy tissue. 161

Figure 5-3. A close view of the identified upregulated proteins (indicated by arrows) altered by the hydrozoan colonization. 162

Figure 5-4. A close view of the identified downregulated proteins (indicated by arrows) found mostly in the healthy tissue but rare in the hydrozoan-colonized tissue. 163

Figure 5-5. A close view of the identified downregulated proteins (indicated by arrows) altered by the hydrozoan colonization. 164

Figure 6-1. Different parts of bryozoans-colonized Saccharina japonica 174

Figure 6-2. Two-DE profiles of the late harvest Saccharina japonica. 215

Figure 6-3. A close view of identified up-regulated proteins (indicated by arrows) in the healthy tissue 1-cm from the lately colonized Saccharina japonica part by Membranipora membranacea. 217

Figure 6-4. A close view of identified down-regulated proteins (indicated by arrows) the healthy tissue 1-cm from the lately colonized Saccharina japonica part by Membranipora membranacea. 218

Figure 6-5. A close view of identified proteins found mostly in the healthy tissue 1-cm from the lately colonized Saccharina japonica by Membranipora membranacea but few in healthy tissue. 219

Figure 6-6. A close view of identified up-regulated proteins (indicated by arrows) in the lately colonized Saccharina japonica by Membranipora membranacea. 220

Figure 6-7. A close view of identified down-regulated proteins (indicated by arrows) the lately colonized Saccharina japonica part by Membranipora membranacea. 221

Figure 6-8. A close view of identified proteins found mostly in the lately colonized Saccharina japonica by Membranipora membranacea but few in healthy tissue. 222

Figure 7-1. Different sections of hydrozoans-colonized Saccharina japonica 231

Figure 7-2. Two-DE profiles of the late harvest Saccharina japonica. 265

Figure 7-3. A close view of identified up-regulated proteins (indicated by arrows) in the healthy tissue 1-㎝ from the lately colonized Saccharina japonica part by Obelia geniculata. 266

Figure 7-4. A close view of identified down-regulated proteins (indicated by arrows) the healthy tissue 1-㎝ from the lately colonized Saccharina japonica part by Obelia geniculata. 267

Figure 7-5. A close view of identified proteins found mostly in the healthy tissue 1-㎝ from the lately colonized Saccharina japonica by Obelia geniculata but few in healthy tissue. 268

Figure 7-6. A close view of identified proteins found mostly in the outer healthy tissue but few in the tissue 1-㎝ from the lately colonized Saccharina japonica by Obelia geniculata. 269

Figure 7-7. A close view of identified up-regulated proteins (indicated by arrows) in the lately colonized Saccharina japonica by Obelia geniculata. 270

Figure 7-8. A close view of identified down-regulated proteins (indicated by arrows) the lately colonized Saccharina japonica part by Obelia geniculata. 271

Figure 7-9. A close view of identified proteins found mostly in the lately colonized Saccharina japonica by Obelia geniculata but few in healthy tissue. 272

Figure 7-10. A close view of identified proteins found mostly in the outer healthy tissue but few in the lately colonized Saccharina japonica by Obelia geniculata. 273

해조류 다시마에 부착하는 기생벌레들의 군체 형성은 해조류 산업에 나쁜 위협이 되고 있으며 양식 기간동안 이들의 부착을 피하는 것은 어려운 일이다. 이같은 부착성 오손 생물들 중 에서 이끼벌레와 혹히드라에 의한 피해가 가장심하다. 따라서 이끼벌레 및 혹히드라군체 형성이 다시마의 생화학적 성분과 다시마 조직의 생존능력에 미치는 영향을 연구하였고 또한 균체 주변의 다시마 세포 단백질체 구성변화도 함께 확인하였으며 마지막으로 군체가 형성된 다시마의 초기 부착 표지 단백질을 식별 하고자 하였다.

우선 다시마의 이끼벌레 자체에서는 회분 (657g/Kg 건조 중량) 및 비소(49 mg/Kg 건조 중량)를 많이 포함하고 있으며 이는 사람의 권장소비범위를 초과하는 높은 수준이었고, 필수 아미노산 함량은 상대적으로 낮은 수준이었다. 비소의 함유량은 세계 보건기구에 의해 설정된 잠정 주간 섭취 허용량 (PTWI)의 31%이였다. 이끼벌레를 제거한 후, 군체 아래의 다시마 조직은 건강한 조직보다 오히려 높은 칼륨, 요오드, DHA 를 함유하였고 구리, 크롬, 카드뮴은 감소한 것으로 나타났다. 마찬가지로, 다시마에 기생하는 혹히드라에는 많은 아연 (1.7 g/kg 건조 중량)이 포함되어있었으며 이는 PTWI 에따른 식품권장범위보다 23% 높은 수준이다. 그리고 혹히드라를 제거한 군체밑의 해조류 조직에서는 건강한 조직에 비해 구리, 카드뮴, 니켈, 크롬 및 erucic acid 농도가 감소 하였고 셀레늄, 요오드, DHA 의 농도는 증가하였다. 따라서 다시마를 식품이나 사료로 사용하는데 있어서는 사용 전에 반드시 해조류 표면에서 제거해야만 한다.

두 번째 연구인 다시마의 단백질체 조성에서는 이끼벌레 군체가 형성된 조직에서는 145 개의 단백질이 검출되었으며 건강한 조직에서는 91 개의 단백질이 검출되었다. 이들 중 69 및 32 개의 단백질들은 이끼벌레에 의하여 유의하게 각각증가 혹은 감소되었다. 이와 유사하게 혹히드라에 감염된 조직과 건강한 조직에서는 75 개와 91 개의 총 단백질 들이 확인되었다. 그 중 77 개와 28 개의 단백질들은 상향 혹은 하향 생성되었다. 상향 또는 하향 조절된 단백질들의 대부분은 스트레스 조절, 방어기작, 신호 전달, 광합성, 단백질 대사와 세포 골격형성에 관련이 있는 것으로 조사되었다.

세번째 연구로서는 이끼벌레 및 혹히드라 감염에 따른 초기 감염 표지 단백질을 찾고자 하였다. 이끼벌레 감염 초기에는 zinc finger protein, 액틴, 키나아제, 티오레독신 도메인 단백질 3 와 ATP 합성 관련 단백질을 함유하는 GAP, GluTR, LRR 과 NB-ARC 도메인, 이성질 반응 조절자, 세린-/피루브산 아미노기 전이 효소, 50S 리보솜 단백질인 L1P, ATP 합성 효소 베타 서브유닛, 케라틴 1 타입인 세포 골격과 접합 인자등이 있다. 마찬가지로, 혹히드라 감염 초기에 발현되는 단백질들은 ATP 합성 베타 서브유닛 (엽록체), ATP 합성 CF1 베타체인(엽록체), 사이토크롬 P450, 이성질 반응 조절자, EXPANSIN, 신호전달증식 연관 단백질 1, 액틴, 단백질 키나아제, 유비퀴틴 전이효소, 아데노신 데아미나제, 키네신 등이 잠재적인 초기 감염 표지 단백질들로 확인되었다.