Author Identifiers

Emily D Lette
ORCID: 0000-0002-0462-8255

Date of Award

2020

Degree Type

Thesis

Degree Name

Master of Science (Biological Sciences) by Research

School

School of Science

First Advisor

Associate Professor Annette Koenders

Second Advisor

Dr Quinton Burnham

Third Advisor

Dr Rodney Duffy

Fourth Advisor

Professor Pierre Horwitz

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.

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 subhypothesis, 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 nontransient; 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.

Access Note

Chapter 4 is not included in this version of the thesis.

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