Application of metabolomics to identify functional metabolic changes associated with Haliotis midae growth
The South African abalone, "perlemoen" (as it is called locally) industry is largely based on farming with Haliotis midae, which has been commercially cultured in man-made shore-based systems with great success for the last 20 years. Due to the basic dynamics of abalone aquaculture being well-known, the high market value and the demand for this delicacy, this sector is commercially, the largest of all aquaculture sectors in SA. However, knowledge of abalone metabolism and the biochemical processes associated with abalone growth and development are lacking. Since maximising growth and health of abalone is the primary goal for optimising production and revenue on abalone farms, research on abalone metabolism could lead to a better understanding of their metabolic responses to specific perturbations and subsequently, to better growth. Metabolomics, one of the newest additions to the "omics" research technologies, aims to investigate the metabolism holistically, and is considered a powerful tool for new biomarker identification and better elucidation of the observed phenotypical changes associated with a perturbation. Considering this, the effects of 1) functional and environmental hypoxia and 2) diet and abalone age as experienced within the standard farming environment, were investigated in Haliotis midae in this thesis. By analysing different tissue samples (adductor muscle, foot muscle, left gill, right gill, haemolymph and epipodial tissue), using a multiplatform (nuclear magnetic resonance spectroscopy, gas chromatography mass spectrometry and liquid chromatography mass spectrometry), standardised metabolomics approach, growth and metabolism of abalone could be elucidated. Univariate statistical methods were used to identify those features of significance, to which metabolite identifiers were assigned, based on well-defined identification guidelines, which were subsequently used in pathway analyses and for biological interpretation of perturbations in relation to growth of abalone. The results show that functional and environmental hypoxia result in a metabolic imbalance in H. midae, with the resulting energy deficit being compensated for by phosphoarginine reserves. This initial response is later supplemented by anaerobic glycolysis, whereby glucose is converted to pyruvate, and then to lactate or opines, in order to replenish the dwindling nicotinamide adenine dinucleotides required as substrates for further adenosine triphosphate production. Furthermore, the metabolomics results also suggest that stressors such as hypoxia, causes abalone to redirect their energy utilisation towards those metabolic pathways essential to the survival of the animal, at the expense of growth. In contrast, the metabolomics analysis done on the adductor muscle samples of abalone, with comparatively good growth rates, showed that faster growing individuals utilise energy pathways and reserves (via elevated insulin production) in such a way that they promote protein synthesis. Furthermore it is suggested that modified artificial abalone feed stimulates mitochondrial function, enabling juvenile abalone to catabolise proline for energy production, while in adult abalone, proline was utilised primarily towards improving energy production through ß-oxidation pathways. From this metabolomics investigation, it becomes evident that abalone have well-developed metabolic mechanisms ensuring survival during periods of oxygen depletion, however, this does inhibit growth, and in the absence of such stress, the metabolism of abalone would favour protein synthesis. At this stage the reasons as to why some individuals utilise amino acid reserves more rapidly for protein synthesis, under the same growth conditions are still debatable. Furthermore, this study proves that metabolomics is an extremely valuable tool for investigating the altered metabolic processes related to growth in abalone, and hence, could be considered a valuable tool for the abalone aquaculture industry, for identifying biomarkers for growth and health monitoring.