There currently exists little specific molecular biology and molecular genetic data about growth and reproduction related processes in abalone. A better understanding of these processes will not only potentially allow growth enhancement and greater control of reproduction in cultured abalone, but will also provide gene sequence data vital to studies of molluscan endocrinology and phylogenetics. Strong inferences about abalone growth and reproduction may be drawn from better studied molluscs. For example, cephalic ganglia from the gastropods Aplysia and Lymnaea are known to produce growth and reproduction affecting neuropeptides.
Here, I use a combination of cDNA- and protein-based approaches to identify and characterise growth and reproduction-related genes produced in the ganglia of the tropical abalone Haliotis asinina, which is a commercially important marine vetigastropod found throughout the South Pacific. First, to identify expressed sequences potentially involved in controlling growth and reproduction, five suppression subtractive hybridisation (SSH) expressed sequence tag (EST) libraries were created from abalone ganglia and foot. Differentially-expressed sequences were assembled, identified, and characterised using a range of bioinformatic tools. Second, gene expression of selected sequences was compared by quantitative reverse transcription-polymerase chain reaction (qPCR) between (1) fast-growing and slow-growing cohorts of juvenile abalone, and (2) anterior ganglia of cohorts of male and female ganglia at intervals throughout the reproductive cycle. Third, protein based methods were used to (1) examine peptide production and secretion from abalone ganglia, (2) examine post-translational processing of secreted peptides, and (3) obtain sequences of putative growth and reproduction related peptides from the haemolymph.
The five SSH libraries produced 784 unassembled cDNA sequences. Assembly resulted in 640 unigenes, of which 139 were identified using bioinformatics tools. Notable genes found in H. asinina for the first time in this study encode molluscan insulin-related peptide (MIP), a schistosomin-like peptide (SLP), the neuromodulator APGWamide, and the previously unknown haliotid growth associated peptide (HGAP). The encoded Has-HGAP and Has-SLP peptides were also sequenced and structurally characterised using proteomic methods. Both of these were found to have structures consistent with being secreted peptides, and the Has-HGAP peptide was also shown to be secreted from combined cerebral and pleuropedal ganglia.
Gene expression was compared between 20 of the fastest growing and 23 of the slowest growing juvenile abalone taken from a population of 500 animals grown in the same artificial culture environment. The Has-HGAP gene was shown to be upregulated in the fast-growing abalone by qPCR. The Has-MIP encoding gene was shown to be growth related, albeit in a manner opposite to literature-derived expectations. Prior literature has nominated MIPs as likely growth-promoting hormones, but my analyses reveal Has-MIP expression to be downregulated in fast-growing abalone. The Has-SLP gene is upregulated in fast-growing animals, and is probably a growth-promoting hormone, consistent with existing knowledge of this gene.
The short, semilunar spawning cycle of H. asinina permits focussed examinations of the entire reproductive cycle. Although wild abalone are known to spawn synchronously, they lose this synchronicity in captivity; their endogenous reproductive cycle time-keeping cues remain unidentified. Further, little is currently known about how the expression of reproduction-affecting genes varies throughout the spawning cycle. Here, I track the expression of established and potential reproduction-related genes throughout the reproductive cycle in order to (1) identify gene expression patterns consistent with a time-keeping cue (2) provide possible insights into gene functions by identifying correspondence between gene expression and known reproductive cycle events, and (3) provide a temporal map of selected gene expression during the reproductive cycle for use in future studies. I show significant Has-APGWamide gene upregulation in female ganglia at time of spawn is consistent with established reproductive roles for this gene. Female expression of Has-Whitnin peaks concurrent with oocyte detachment from the gonad wall. The expression of Has-FMRFamide and Has-SLP show expression profiles consistent with conserved roles in the inhibition of female reproductive processes; Has-FMRFamide expression may reflect a time-keeping cue 36 h prior to spawning.
This thesis has resulted in the isolation and characterisation of the novel secreted peptide Has-HGAP, the schistosomin-resembling hormone Has-SLP, and their encoding genes. The classical growth related gene Has-MIP is also identified, as are the classical reproduction related, neuromodulator-encoding genes Has-APGWamide and Has-Myomodulin, and finally the newly characterised Has-Whitnin. The differing expression of Has-HGAP, Has-MIP, and Has-SLP between fast and slow-growing abalone identifies these genes as potentially useful markers for growth rate, although the downregulation of Has-MIP in fast-growing abalone may suggest an unexpected role for this gene in growth inhibition. The distinctive expression profile shown by neuromodulators throughout the reproductive cycle suggests that they contribute to controlling this process, and may be modulated in the hatchery. Thus, the neuropeptide genes characterised here may assist in abalone broodstock selection, reproductive cycle regulation, and future genetic studies of growth and reproduction.