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研究生:邱于哲
研究生(外文):Yu-Che Chiu
論文名稱:探討表皮黏膜微生物於含氨水體及虎頭鯊鯰魚表皮之間所扮演的角色
論文名稱(外文):Investigation into the role of skin mucus microbiome in the interplay between environmental ammonia and striped catfish (Pangasianodon hypophthalmus) skin
指導教授:王亮鈞王亮鈞引用關係湯森林
指導教授(外文):Wang,Liang-ChunTang,Sen-Lin
學位類別:碩士
校院名稱:國立中山大學
系所名稱:海洋生物科技暨資源學系研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2024
畢業學年度:112
語文別:英文
論文頁數:116
中文關鍵詞:魚表皮魚表皮黏膜表皮黏膜微生物氨攻毒試驗氨利用菌
外文關鍵詞:Fish skinFish skin mucusSkin mucus microbiomeAmmonia challengeAmmonia-utilizing bacteria
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氨是一種常見於水生環境、對於水生生物的健康具有威脅性的含氮汙染物。在養殖產業中,高密度的養殖容易因管控不當造成氨在水中累積而危害魚隻。然而,魚表皮作為水生環境與魚體之間最主要的外部組織,卻鮮少研究探討其如何應對環境中的氨。因此,本篇研究以養殖魚種虎頭鯊鯰魚(Pangasianodon hypophthalmus)為實驗對象,旨在評估魚表皮如何應對環境中的氨,同時探討魚表皮外的黏膜層是否在氨引起魚表皮的反應之間扮演著關鍵角色。透過表皮黏膜有無被移除的同時進行氨攻毒實驗,我們發現環境中的氨確實會傷害表皮組織,例如外表皮層的剝離及發炎反應,且證實了黏膜的存在可以減少氨引起的傷害。透過將表皮黏膜培養在富含氨的培養基,我們揭示了黏膜中存在利用氨的細菌,例如Acinetobacter sp.,推測其能減緩魚表皮受氨的傷害。透過16S rRNA gene序列以及Ecoplate培養分析表皮黏膜微生物,我們發現當氨濃度超過表皮黏膜減緩氨傷害的能力時,表皮黏膜微生物的組成及代謝功能會改變。綜上所述,本篇研究揭示了在表皮黏膜微生物的參與之下,環境中的氨與魚表皮之間的複雜關係,藉由了解魚表皮及其黏膜微生物如何應對富含氨的環境,希望在未來發展更成熟管控魚體健康的方法,實際應用於養殖現場,促進養殖產業的成長。
Ammonia is a well-known aquatic nitrogenous pollutant and poses a threat to fish health, particularly in aquaculture. Fish skin with the mucus and residential microbiome serves as the primary external interface between fish and the aquatic environment; however, its responses to ammonia remain unexplored. The present study thereby evaluated the skin responses of striped catfish (Pangasianodon hypophthalmus) to environmental ammonia, as well as investigated the potential role of skin mucus in intervening ammonia-induced effects. We found that ammonia can directly damage fish skin tissues by inducing epidermal exfoliation along with inflammation, yet the skin mucus could reduce this damage, as demonstrated through a skin mucus removal approach. Cultivation of the skin mucus in an ammonia-enriched medium revealed the presence of ammonia-utilizing bacteria, especially Acinetobacter sp., within the skin mucus microbiome, suggesting a potential mitigation capacity against ammonia. Skin mucus microbiome analysis using 16S rRNA gene sequences and metabolic function evaluation by Ecoplate assay indicated skin microbiome shifts while ammonia presents beyond the mitigation capacity of skin mucus. Our work sheds light on the complex interplay between environmental ammonia and host skin, under skin microbiome’s intervention. Understanding how fish skin and its mucus microbiome respond to ammonia-enriched environments will help develop a better aquaculture practice in monitoring fish health.
論文審定書 i
誌謝 ii
摘要 iv
Abstract v
Table of Contents vii
List of Figures xi
List of Tables xiii
Abbreviations xiv
1 Introduction 1
1.1 Aquaculture 1
1.1.1 Aquaculture consumption and nitrogen-based issues 1
1.1.2 Niches of striped catfish 3
1.3 Fish skin and skin mucus microbiome 6
1.4 Factors that can modulate fish skin microbiome 8
1.5 Impact of ammonia on the microbiome 10
1.6 Ammonia removal by bacteria in aquatic environments 11
1.7 Aims of the present study 13
2 Materials and Methods 14
2.1 Materials 14
2.1.1 Experimental animal 14
2.1.2 Equipment and suppliers 15
2.2 Methods 16
2.2.1 Fish husbandry and water quality measurement 16
2.2.2 Skin mucus removal approach 16
2.2.3 Ammonia challenge along with skin mucus removal in fingerlings 17
2.2.4 Different concentrations of ammonia challenge in juveniles 18
2.2.5 Histological examination of skin tissues 19
2.2.6 Skin tissue ammonia measurement 19
2.2.7 Total RNA extraction of fish skin and Real-Time PCR analysis 20
2.2.8 Skin mucus microbial DNA preparation 21
2.2.9 16S rRNA gene sequencing and microbiome analysis 22
2.2.10 Biolog Ecoplate inoculation and measurement 23
2.2.11 Assessment of ammonia-utilizing capacity 24
2.2.12 Examination of potential ammonia-utilizing bacteria 25
2.2.13 Statistical analysis 26
3 Results 28
3.1 Skin mucus removal approach in the present study 28
3.1.1 Skin mucus can be efficiently removed using soft tissue paper 28
3.1.2 Skin mucus is removed without discernible damage to the skin 30
3.2 Skin mucus mitigates the ammonia damage in fish skin tissue potentially through reducing tissue ammonia accumulation 32
3.3 Fish skin with mucus is minimally damaged or undamaged under ammonia challenge concentration that can induce the health burden 39
3.4 The microbial community in fish skin mucus shifts under ammonia challenge 43
3.5 Metabolic functionality of skin mucus microbiome is altered corresponding to microbial composition shift under ammonia challenge 49
3.6 Carbon- and nitrogen-based metabolism of viable skin mucus microbiome increases by ammonia challenge 53
3.7 The skin mucus microbiome can utilize ammonia for growth 56
3.8 Isolated Acinetobacter sp. in the skin mucus microbiome contributes to ammonia utilization 59
3.9 Compositional loss of Acinetobacter sp. in the skin microbiome after mucus removal with or without ammonia challenge 62
4 Discussion 64
4.1 Summary 64
4.2 Mucus removal approach in fish skin 64
4.3 The damage of ammonia on fish skin 66
4.4 Mitigation function of fish skin mucus against ammonia 68
4.5 The role of mucus microbiome on fish skin in response to ammonia 72
4.6 Microenvironment on fish skin under the presence of ammonia 78
4.7 Limitations and Future Perspectives 83
4.8 Prospective Model and Conclusion 85
5 References 88
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