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A Metabolomic Investigation of a Critically Endangered Freshwater Crayfish

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A Metabolomic Investigation of a Critically Endangered Freshwater Crayfish Edith Cowan University Research Online Theses: Doctorates and Masters Theses 2020 Modernising Conservation Through Technology: A metabolomic investigation of a critically endangered freshwater crayfish Emily D. Lette Edith Cowan University Follow this and additional works at: https://ro.ecu.edu.au/theses Part of the Marine Biology Commons Recommended Citation Lette, E. D. (2020). Modernising Conservation Through Technology: A metabolomic investigation of a critically endangered freshwater crayfish. https://ro.ecu.edu.au/theses/2371 This Thesis is posted at Research Online. https://ro.ecu.edu.au/theses/2371 Edith Cowan University Copyright Warning You may print or download ONE copy of this document for the purpose of your own research or study. The University does not authorize you to copy, communicate or otherwise make available electronically to any other person any copyright material contained on this site. You are reminded of the following: Copyright owners are entitled to take legal action against persons who infringe their copyright. A reproduction of material that is protected by copyright may be a copyright infringement. Where the reproduction of such material is done without attribution of authorship, with false attribution of authorship or the authorship is treated in a derogatory manner, this may be a breach of the author’s moral rights contained in Part IX of the Copyright Act 1968 (Cth). Courts have the power to impose a wide range of civil and criminal sanctions for infringement of copyright, infringement of moral rights and other offences under the Copyright Act 1968 (Cth). Higher penalties may apply, and higher damages may be awarded, for offences and infringements involving the conversion of material into digital or electronic form. Modernising Conservation Through Technology: A metabolomic investigation of a critically endangered freshwater crayfish This thesis is presented in fulfilment of the requirements for the degree of Master of Science (Biological Sciences) by Research Emily D. Lette B.Sc. Biological Sciences (Conservation Biology) Supervisors: Associate Professor Annette Koenders, Dr. Quinton Burnham, Dr. Rodney Duffy, and Professor Pierre Horwitz School of Science Edith Cowan University 2020 USE OF THESIS The Use of Thesis statement is not included in this version of the thesis. Abstract BACKGROUND: The Anthropocene has seen extinction rates orders of magnitude higher than the background rate; a trend that has been seen in all ecosystems. As a result of human activities, freshwater resources, and many of the species dependent on them have become imperilled. Freshwater crayfish are a dominant aquatic invertebrate due to their significant biomass, and they are globally distributed, highly speciose, and ecologically important. They have been referred to, inter alia, as bioindicators, keystone species, ecosystem engineers, and umbrella species and are also a valuable human food source. However, currently one-third of freshwater crayfish species worldwide are classified as threatened under IUCN criteria, with many species facing possible extinction. Cherax tenuimanus (hairy marron) is a critically endangered freshwater crayfish found only in a single river in the biodiversity hotspot of south-west Australia. Conservation efforts for this species have included a captive breeding program, which has been largely unsuccessful despite the successful breeding of sister taxon Cherax cainii (smooth marron) for aquaculture. Currently captive breeding, including aquaculture of crayfish, relies primarily upon traditional methods of investigating the impacts of environmental factors through gross trial and error, with little understanding of the physiological state of animals. This study tested the hypothesis that metabolomics could highlight potential biomarkers related to reproduction and stress in two congeneric freshwater crayfish, Cherax tenuimanus and Cherax cainii, for the purpose of providing information to assist with captive breeding. HYPOTHESIS TESTING: In order to test this hypothesis, four sub-hypotheses were tested in this study. Sub-hypothesis I: C. tenuimanus can be induced to breed in aquaria. This hypothesis was supported, as mating occurred in both species of marron. Timing of reproductive behaviours was later in C. tenuimanus and fecundity was lower than C. cainii. Breeding behaviours were documented in detail. Sub-hypothesis II: The reproductive hormone methyl farnesoate (MF) can be measured in marron haemolymph as a non-lethal, low stress tool to monitor reproduction (i.e. as a targeted metabolomic approach). This hypothesis could not be confirmed or rejected, because MF was not detected using two extraction methods. Sub-hypothesis III: Untargeted metabolomics using liquid chromatography–mass spectrometry (LC-MS) detects differences in the metabolome between species and sexes of marron. The profiles of C. tenuimanus and C. cainii were significantly different, as were the profiles between the sexes of each species. iii Sub-hypothesis IV: Untargeted metabolomics using LC-MS detects differential responses in C. tenuimanus females and males in breeding pairs. The metabolite profiles supported this sub- hypothesis, where three patterns were identified by the behaviour of the metabolites. Metabolites either indicated a response to disturbance (change) where the response was transient or non- transient; differences between sexes where the differences remained unchanged whether the animals were housed on their own or with a potential mate; or a male response to female presence. Metabolites such as inosine, glutathione and arginine were recognised as potentially useful biomarkers. CONCLUSIONS: This study demonstrates that metabolomics are useful in providing an informative profile and identifying biomarkers that have the potential to assist with the captive breeding of freshwater crayfish. Whilst a single metabolite (MF) could not be directly targeted in this study, an untargeted approach was successful, and by extension the overall hypothesis of this study was successful. Overall, 107 metabolites were detected in marron haemolymph: amino acids, lipids, nucleotides, and other compounds were successfully linked to biologically important processes in the marron life cycle. The metabolites identified by this approach showed differences between two congeneric species, between sexes and over time in response to an environmental stressor. The study highlighted potential biomarkers for targeted metabolomic studies that can be used to test a wide variety of hypotheses, especially when animals are kept in controlled conditions such as in this study. The investigations from this study also contribute to our understanding of the life history of C. tenuimanus, our knowledge of its reproductive biology and the differences with its sister species C. cainii, providing another piece to the conservation puzzle. These methods will be beneficial to species conservationists and aquaculturists alike. iv Declaration I certify that this thesis does not, to the best of my knowledge and belief: i. incorporate without acknowledgment any material previously submitted for a degree or diploma in any institution of higher education; ii. contain any material previously published or written by another person except where due reference is made in the text of this thesis; or iii. contain any defamatory material. iv. contain any data that has not been collected in a manner consistent with ethics approval. Signed: Emily D. Lette Date: 31 March 2020 Co-authorship statement This thesis contains one manuscript titled ‘Metabolomic profiling of crayfish haemolymph: an investigation of differences between marron species and sexes’ located in Chapter 4 and will be published in the journal Freshwater Crayfish 25(1), 2020. Emily D. Lette is the first author because of her role in contributing to the planning, preparation and execution of the research project and subsequent paper. Dr. Nathan Lawler is second author due to his contribution to the metabolomics analysis and interpretation of the research data. Dr. Quinton Burnham is a co-author as he supervised all aspects of the research and for contributions to editing the paper. For their expertise and provision of funding, Associate Professor Mary Boyce and Dr. Rodney Duffy are co-authors. The last authors are Associate Professor Annette Koenders who was co-supervisor for the research and Professor David Broadhurst who provided metabolomics analysis and statistical support; both had a role in identifying the research objective for this paper. v Publications arising from this research Journal articles: Lette, E. D., Lawler N. G., Burnham, Q. F., Boyce, M. C., Duffy, R., Koenders, A., Broadhurst, D. I., (2020) Metabolomic Profiling of Crayfish Haemolymph Distinguishes Sister Species and Sex: Implications for Conservation, Aquaculture and Physiological Studies. Freshwater Crayfish. 25 (1) Conference: Reinke, S. N., Tu Y., Lawler N., Lette, E., Koenders, A., Boyce, M. (2017) Metabolomics in Undergraduate Education – A Workflow for Authentic Learning. [Abstract and poster presentation] MetaboMeeting 2017, Birmingham, UK, 11-13 Dec 2017 Lette, E., Koenders, A., Burnham Q., Duffy R., Horwitz, P. (2018) Profiling the Metabolome of two Cherax species to investigate their Reproductive Physiology. [Abstract and oral presentation] International Association of Astacology 22nd International Symposium on Freshwater Crayfish, Pittsburgh, USA, 9-13th July 2018. Lette, E. (2019). Metabolomic profiling of
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