Metabolomics Applications in Immunological Studies of Marine Molluscs
Nguyen, Van Thao
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Molluscs form an important group in aquaculture as well as in coastal wild systems. However, high mortalities in molluscan species, specifically marine bivalves, have been encountered in the wild during summer times (summer mortality) as well as in aquaculture settings, which present a major economical challenge in many parts of the world. The complex interactions between host, environment and pathogens during these mortality events require new diagnostic tools and integrated approaches. Metabolomics is one of the newest and fastest growing omics. The sensitivity and specificity of metabolomics approaches make this a powerful tool for immunological studies, where it can provide insights into disease processes as well as the identification of metabolite biomarkers for early warning systems. This thesis was designed to provide, for the first time, a comprehensive understanding of the metabolic responses of mussel haemocytes and other tissues (e.g., gills, hepatopancreas, mantle) to external stimuli (Vibrio sp., lipopolysaccharides [LPS], Cu²⁺) using gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach. Along with the core metabolomics tool, novel flow cytometry (FCM) protocols were developed in order to assess immunological parameters of the host upon stimulation. The combined method allows characterization of the mussel immune responses at both cellular and molecular levels and expands the number of biomarkers used to understand the animal’s response. Initially, tissue-specific metabolic responses of gill, haemolymph and hepatopancreas were observed in mussels challenged with Vibrio sp. Then, haemolymph was chosen as the target tissue/organ for the rest of the experiments in the thesis (Chapter 4). FCM revealed sex-based differences in immune responses of mussels to Vibrio sp. challenge. In this case, female mussels had lower haemocyte mortality, production of reactive oxygen species (ROS) and apoptotic cells after pathogen exposure compared to male mussels (Chapter 5). This suggests that female mussels have more efficient defence system than male mussels. However, metabolite profiles of haemolymph showed no significant difference between males and females. Subsequently, metabolic profiles of mussel haemolymph were intensively investigated in response to Vibrio sp. challenge, LPS and copper exposure (Chapter 6, 7 & 8). The alterations of metabolite profiles along with changes in immune characteristics due to stimulation provided insights into a number of pathways involved in immune responses of the host to Vibrio sp. infection and copper exposure. The study also identified a number of candidate biomarkers involved in mussel immune processes. Among these metabolites, the presence of itaconic acid (ITA) and its accumulation were observed in different tissues of mussels following Vibrio sp. challenges, suggesting the important role of this metabolite as an antimicrobial compound in the innate immune system of bivalves (Chapter 4, 5 & 6). In fact, the challenge experiment (Chapter 9) revealed the complete inhibition of ITA on Vibiro sp. growth at 6 mM, and Vibrio growth was partially inhibited at 3 mM ITA. This confirmed, for the first time, the antibacterial activity of ITA against marine Vibiro sp. and suggests that ITA could be used as an antimicrobial compound for antibiotic resistant bacteria in aquaculture. Subsequently, the ITA concentrations in different tissues of mussels challenged with Vibrio sp. were quantitatively measured (Chapter 10). Interestingly, the results revealed that mussels are able to produce an effective amount of ITA to support the internal defence system, suggesting that ITA could be a valuable biomarker for health assessment of bivalves. In addition, ITA may also involve in anti-inflammation activities and other unknown functions in the bivalve innate immune system, which need further studies to reveal. In conclusion, this thesis has successfully demonstrated the use of novel metabolomics approaches for aquaculture and marine science, which contribute new information regarding the molluscan immune system. It is envisaged that metabolomics will continue to grow as a tool of choice in studies of marine molluscs, as well as the broader field of marine science.