Author: Omar Mendoza Porras
Mendoza Porras, Omar, 2014 Molecular assessment of spawning cues in temperate abalone Haliotis laevigata, Flinders University, School of Biological Sciences
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Abalone are edible marine snails distributed throughout the world in tropical and temperate waters. Abalone are highly valued for their palatability, particularly in Asian countries where consumption is inherent to traditions. Wild caught abalone supplies some of the global market, however to meet increasing commercial demand abalone are also farmed. In fact, the majority of abalone supplied on the global market is produced on farms. Despite the advances in abalone farming, many issues remain unresolved such as prolonged feeding requirements, disease management and inefficient spawning all of which have downstream affects on production. In temperate greenlip abalone Haliotis laevigata, inefficient spawning has limited pair-wise breeding of selected broodstock that would otherwise produce highly marketable abalone. Understanding the mechanisms that control spawning and identifying the molecule(s) associated with abalone spawning are key to solving this issue. This research utilised proteomics coupled to mass spectrometry (MS) to profile the gonad proteome in sexually mature abalone and further to identify proteins that were differentially expressed in gonads during artificially induced spawning. Mass spectrometry spectral datasets were searched against two publicly available molluscan databases (UniProt and NCBI) and a custom-built database comprising genomic information from closely related molluscan species to profile the gonad proteome. The results from the multi-database approach were collated and protein redundancy manually removed. A total of 110 and 162 proteins were identified in males and females respectively. From those proteins, 47 were involved in sperm and egg structure, sexual maturation, acrosomal reaction and fertilisation. Utilisation of the custom-built database increased the protein identification rate by more than 60% compared to searches employing public protein databases alone. To identify differentially expressed proteins, male and female abalone gonads from different physiological states (spawning, post-spawning and failed-to-spawn) were studied using two-dimensional difference in gel electrophoresis coupled to MS. The gonads were compared as follows: spawning versus failed-to-spawn; spawning versus post-spawning and post-spawning versus failed-to-spawn. Proteins from these gonads were fluorescently labelled and separated according to isoelectric point and relative molecular weight. The change in protein expression level was assessed for the three pair-wise comparisons facilitating the location of differentially expressed proteins. The selected gel spots were excised and identified using tandem MS. The corresponding MS/MS datasets were searched against UniProt and NCBI protein databases. Gene ontology of the identified proteins revealed roles in gametogenesis, egg and sperm structure, acrosomal reaction, fertilisation, energy metabolism and oxidative stress. The differentially expressed proteins were quantified by multiple reaction monitoring mass spectrometry (MRM-MS) across all physiological states. Significant differences in the abundance of reproductive and anti-oxidant proteins were observed across the physiological states. These differences correlated with propensity or failure to spawn. For example the reproductive protein vitelline receptor for lysin (VERL) was significantly higher in gonads from spawning abalone relative to gonads from post-spawning abalone when assessed in female abalone. In males, sperm protein, atrial natriuretic peptide receptor A (ANF) and chaperonin containing tcp1 (CCT) showed significant differences in gonads from spawning abalone in comparison to gonads from post-spawning abalone. Additionally, significant differences in abundance of antioxidant proteins were observed in both abalone sexes. For female and male ferritin was rendered significantly different in gonads from post-spawning abalone compared to both spawning and failed-to-spawn abalone. Superoxide dismutase (SOD) and peroxiredoxin 6 (Prx6) were significantly different in gonads of spawning and post-spawning of female abalone, whereas thioredoxin peroxidase 2 (TPx2) was significantly different in male gonads of spawning compared to post-spawning abalone. Overall, the majority of significant differences were detected when gonads from spawning and post-spawning abalone were compared. This research revealed the identity of proteins differentially expressed during spawning that may prove to be key molecules involved in H. laevigata spawning. The combined findings of this research provide a greater understanding of the protein expression in abalone gonads following artificial induction of spawning. This fundamental knowledge will inform future research programs that aim to develop new techniques to improve artificial spawning rates on farms. This research also provides the identity of proteins that will assist in performing accurate assessments of sexual maturity in selected abalone broodstock to better predict spawning propensity. In conclusion, genomic and proteomics tools have been used to perform a comprehensive study of abalone gonad protein composition, revealing protein identities and enabling measurement of protein expression levels in abalone gonads following artificial spawning induction.
Keywords: Abalone spawning, proteomics, acrosomal reaction, fertilisation, mollusc 2D-DIGE, Haliotis, mass spectrometry
Subject: Aquaculture thesis, Biological Sciences thesis, Biology thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Dr James Harris