Metabolomic Applications in Marine Mollusc Development and Aquaculture

395 Pages·2017·6.7 MB·English

Metabolomic Applications in
Marine Mollusc Development
and Aquaculture
Tim Young
A thesis submitted to Auckland University of Technology in fulfilment of
the requirements for the degree of Doctor of Philosophy
(PhD)
Faculty of Health and Environmental Sciences
School of Science
2016
Abstract
Metabolomics is a rapidly emerging discipline within functional genomics to better
understand biochemical phenotypes across a range of biological systems. The approach
has many demonstrated applications in aquatic biology, but has not yet been applied to
study early lifestages of marine molluscs. This Thesis evaluates metabolomics as an
approach to characterise early lifestage phenotypes of molluscs, and demonstrates unique
applications in aquaculture, developmental biology, immunology, and toxicology.
GC/MS-based metabolomics was first tested for its capacity to classify good and
bad quality mussel larvae (i.e., slow- vs fast-growing organisms). Based on the
composition of metabolites, larval classes could clearly be discriminated and the data
indicated differences energy metabolism, osmotic regulation, immune function and cell–
cell communication.
Mussel larvae which had been subjected to handling stress and different culture
conditions were also assessed. A decrease in succinate and an increase in alanine were
observed after the water exchange, which indicated alterations in energy production and
osmotic balance. However, these variations were subtle and it is unlikely that the water
exchange practice had any lasting negative effects on larval physiology and performance.
A culture condition classification model was also constructed which revealed that larvae
from flowthrough vs static systems differed in terms of energy, protein and lipid
metabolism. The data also suggests that growth performance is metabolically buffered
through an adaptive physiological mechanism to provide similar developmental
characteristics under these conditions.
Oyster larvae were assessed during a viral (OsHV-1 µVar) infection to characterise
the host-virus interaction at a metabolic level. Responses included a coordinated
disruption of the TCA cycle in accordance with mammalian macrophage stimulation via
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activation of immunoresponsive gene 1 and production of itaconic acid, induction of a
Warburg-like effect, and production of free fatty acids for virion assembly, among others.
These results provide new insights into the pathogenic mechanisms of OsHV-1 infection
in oyster larvae, which may be applied for selective breeding programmes aiming to
enhance viral resistance.
Lastly, metabolomics was applied to investigate mechanisms of toxicity in mussel
larvae exposed to copper contamination. At sublethal dose levels, metabolic trajectory
analysis indicated that larvae were successfully employing various endogenous
mechanisms involving biosynthesis of antioxidants and a restructuring of energy-related
metabolism in an attempt to alleviate the toxic effects on cells and developing tissues.This
was partly confirmed by a targeted analysis of oxidative stress biomarkers (e.g.,
enzymes). A lethal copper dose induced severe metabolic dysregulation after 3 hrs
exposure which worsened with time, substantially delayed embryonic development,
initiated the apoptotic pathway, provided many evidences for the occurrence of oxidative
stress (validated via oxidative stress biomarkers), and resulted in cell/organism death
shortly after 18 hrs exposure.
In summary, this Thesis provides strong support for the application of
metabolomics to assess the health status of marine mollusc embryos and larvae.
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Table of Contents
Abstract ……………………………………………………………………………… i
Table of Contents ……………………………………………………………………. iii
List of Tables ………………………………………………………………………... ix
List of Figures ………………………………………………………………………. x
Attestation of Authorship …………………………………………………………… xiv
Co-author contributions …………………………………………………………….. xv
Acknowledgements …………………………………………………………………. xviii
CHAPTER 1 – SCOPE
Thesis introduction and framework ………………………………………….. 1

1.1 General introduction …………………………………………………… 2
1.1.1 Mollusc aquaculture …………………………………………...... 3
1.1.2 Early lifestage molluscan research ……..……………………….. 7
1.1.3 Moving forward ...………………………………………………. 11
1.2 Thesis motivation ……………………………………………………… 12
1.3 Thesis aims ……………………………………………………………. 15
1.4 Thesis structure ………………………………………………………... 15
1.5 Chapter contents and rationales .……………………………………..... 16
1.6 Research outputs ………………………………………………………. 21
1.6.1 Conference presentations ………………………………………. 21
1.6.2 Industry publications …………………………………………… 22
1.6.3 Journal publications ……………………………………………. 23
1.7 References …………………………………………………………….. 24
CHAPTER 2 – LITERATURE REVIEW 1
Metabolomic applications in aquaculture …………………………………… 32
2.1 Global aquaculture …………………………………………………….. 34
2.2 ‘Omics’ approaches …………………………………………………… 34
2.3 What is metabolomics? ……………………………………………….. 38
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2.4 Advantages of metabolomics …………………………………………. 39
2.5 Metabolomic research …………………………………………………. 40
2.6 Metabolomics applications in aquaculture research …………………… 41
2.6.1 Hatchery production ……………………………………………. 41
2.6.2 Nutrition and diet ...…………………………………………….. 43
2.6.3 Disease and immunology ………………………………………. 46
2.6.4 Post-harvest product quality ……………………………………. 51
2.7 Future applications and directions …………………………………….. 54
2.8 Conclusions ……………………………………………………………. 57
2.9 References ……………………………………………………………... 58

CHAPTER 3 – LITERATURE REVIEW 2
Metabolomic strategies for aquatic research ……………………………….. 68

3.1 Introduction …………………………………………………………… 70
3.2 Metabolomic Strategies ………………………………………………. 71
3.2.1 Experimental design and sampling ……………………………. 72
3.2.2 Analytical platforms …………………………………………… 76
3.3 Metabolite fingerprinting vs. profiling ………………………………... 93
3.4 Data analysis …………………………………………………………... 94
3.4.1 Univariate methods …………………………………………….. 96
3.4.2 Multivariate methods …………………………………………... 98

3.5 Biomarker discovery and validation ………………………………….. 104

3.6 Biological interpretation and secondary bioinformatics ……………… 106

3.7 Reporting guidelines in metabolomics ………………………………... 113
3.8 Incorporating metabolomics …………………………………………... 115

3.9 Summary ……………………………………………………………… 116

3.10 References …………………………………………………………… 117

CHAPTER 4 – CASE STUDY 1
Quality assessment of hatchery-reared mussel larvae ……………………... 133

4.1 Introduction ........................................................................................... 135
4.2 Methods ………………………………………………………………. 137
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4.2.1 Broodstock spawning ………………………………………….. 137
4.2.2 Gamete collection and fertilisations …………………………… 138
4.2.3 Larval production and sampling ……………………………….. 139
4.2.4 Metabolite extraction and derivatisation ………………………. 139
4.2.5 GC-MS analysis ……………………………………………….. 141
4.2.6 Data pre-processing and metabolite identification ……………. 141
4.2.7 Statistical analyses …………………………………………….. 142
4.3 Results ………………………………………………………………... 145
4.4 Discussion ……………………………………………………………. 148
4.5 Conclusion …………………………………………………………… 150
4.6 References ……………………………………………………………. 152

CHAPTER 5 – CASE STUDY 2
Effect of handling and culture conditions on mussel larvae ………………… 156

5.1 Introduction …………………………………………………………... 158
5.2 Methods ………………………………………………………………. 161
5.2.1 Larval rearing ………………………………………………….. 161
5.2.2 Larval sampling ………………………………………………... 161
5.2.3 Metabolomics ………………………………………………….. 163
5.2.4 Statistical analyses ……………………………………………... 163
5.3 Results ………………………………………………………………… 165
5.3.1 Effect of handling and water exchange ………………………… 165
5.3.2 Effect of culture system ………………………………………... 167
5.4 Discussion ……………………………………………………………... 171
5.5 Conclusion …………………………………………………………….. 174
5.6 References ……………………………………………………………... 175

CHAPTER 6 – CASE STUDY 3
Host-virus interactions in oyster larvae …………………………………….... 178

6.1 Introduction ……………………………………………………………. 180
6.2 Methods ……………………………………………………………….. 185
6.2.1 OsHV-1 µVar preparation ……………………………………... 185
6.2.2 Larval production ………………………………………………. 186

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6.2.3 Viral challenge and larval sampling …………………………… 187
6.2.4 Metabolite extraction and GC-MS analysis …………………… 188
6.2.5 Spectral processing and metabolite identification …………….. 188
6.2.6 Statistics ……………………………………………………….. 189
6.3 Results ………………………………………………………………… 194
6.3.1 Univariate analysis …………………………………………….. 194
6.3.2 Unsupervised multivariate cluster analysis ……………………. 195
6.3.3 Supervised multivariate classification analysis ………………... 197
6.3.4 Functional biochemical pathway analysis …………………….. 200
6.3.5 Correlation analysis ……………………………………………. 203
6.4 Discussion …………………………………………………………….. 205
6.4.1 Lipid metabolism ………………………………………………. 206
6.4.2 TCA cycle and immunoresponsive gene 1 …………………….. 212
6.4.3 Warburg effect …………………………………………………. 217
6.4.4 Oxidative stress ………………………………………………… 219
6.4.5 Other signatures ………………………………………………... 221
6.5 Conclusion …………………………………………………………….. 225
6.6 References …………………………………………………………….. 227
CHAPTER 7 – CASE STUDY 4
Copper toxicity during mussel embryogenesis ……………………………… 245
Part A: Untargeted metabolite profiling ………………………………… 246
7.1 Introduction ……………………………………………………… 248
7.2 Methods ………………………………………………………….. 252
7.2.1 Experimental design summary …………………………… 252
7.2.2 Chemicals and seawater preparation ……………………... 252
7.2.3 Broodstock collection and spawning …………………….. 254
7.2.4 Fertilisation and tank incubation …………………………. 254
7.2.5 Embryo and larval sampling ……………………………… 254
7.2.6 Seawater chemistry ………………………………………. 256
7.2.7 Metabolite analysis ………………………………………. 258
7.2.8 Statistical analysis and data visualisation ………………... 258
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7.3 Results …………………………………………………………… 262
7.3.1 Seawater chemistry ………………………………………. 262
7.3.2 Survival and Development ……………………………….. 263
7.3.3 Metabolomics …………………………………………...... 265
7.4 Discussion ……………………………………………………….. 276
7.4.1 Bulk seawater composition ………………………………. 276

7.4.2 Cu speciation, toxicity and macroscopic endpoints ……… 276
7.4.3 Metabolomics …………………………………………….. 279
7.4.4 Metabolomics as a health assessment tool ……………….. 302
7.5 References ……………………………………………………….. 304
Part B: Targeted analysis of oxidative stress biomarkers ………………. 324
7.6 Introduction ……………………………………………………… 326
7.7 Methods ………………………………………………………….. 329
7.7.1 Experimental design summary and sampling …………….. 329
7.7.2 Protein, lipid and DNA analysis ………………………….. 329
7.7.3 Antioxidant enzyme analysis ……………………………... 332
7.7.4 Reduced glutathione analysis …………………………....... 334
7.7.5 ROS analysis ……………………………………………… 334
7.7.6 Statistics and data presentation …………………………… 335

7.8 Results …………………………………………………………… 336
7.9 Discussion ……………………………………………………….. 340
7.9.1 Reactive oxygen species ………………………………….. 340
7.9.2 Glutathione ………………………………………………... 342
7.9.3 Enzymatic biomarkers of oxidative stress ………………… 343
7.9.4 Macromolecular biomarkers of oxidative damage ………... 347
7.10 Conclusion ……………………………………………………… 348
7.11 References ……………………………………………………… 350
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CHAPTER 8 – SYNTHESIS
Discussion and conclusions ………………………………………………….. 356
8.1 Thesis background …………………………………………………… 357
8.2 Core chapter philosophies …………………………………………… 359
8.3 Study limitations …………………………………………………….. 367
8.4 Future metabolomics research ………………………………………. 369
8.5 Conclusion …………………………………………………………… 374
8.6 References ……………………………………………………………. 375
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List of Tables
Table 3.1 A selection of studies using metabolomics-based approaches with
relevance to aquaculture ……………………………………………. ….. 80
Table 3.2 Comparisons between different analytical platforms for processing
metabolomics samples ……………………………………………... ….. 92
Table 3.3 Summary of sample-specific topics which should be described in
detail when reporting the results of a metabolomics project ………... ….. 114
Table 4.1 Degree of importance and rank frequency of candidate biomarkers
in models for larval class separation ……………………………….. ….. 147
Table 4.2 Candidate biomarker ratios and associated relative metabolite
abundances in high and poor quality larvae ………………………... ….. 147
Table 5.1 Larval rearing parameters used during low-density static and high-
density flow through culture ……………………………………….. ….. 162
Table 5.2 Metabolic profiles of larvae before and after a prolonged handling
and water exchange process ………………………………………… ….. 165
Table 5.3 Metabolic profiles of larvae reared to ten days post-fertilisation in
low-density static and high-density flow through systems ………… ….. 166
Table 5.4 Statistical analyses of metabolite ratio biomarkers for assessing
quality of larvae reared in different culture systems ……………….. ….. 174
Table 6.1 List of altered metabolic pathways in larval hosts during viral
(OsHV-1 μVar) infection …………………………………………... ….. 204
Table 7.1 Composition of the bulk seawater used (prior to EDTA additions)
during a copper stress experiment ………………………………….. ….. 262
Table 7.2 Copper speciation in seawater (after EDTA and CuSO additions)
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during a copper stress experiment ………………………………….. ….. 262
Table 7.3 Targeted analysis of oxidative stress biomarkers in mussel embryos
and larvae in response to copper exposure …………………………. ….. 338
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