| Summary: | Thesis (PhD (Genetics))--University of Stellenbosch, 2010. === Includes bibliography. === ENGLISH ABSTRACT: The slow growth rate of Haliotis midae impedes the optimal commercial production of this most
profitable South African aquaculture species. To date, no comprehensive effort has been made to
identify genes associated with growth variation in farmed H. midae. The aim of this study was therefore
to investigate growth variation in H. midae and to identify and quantify the expression of selected
growth-related genes. Towards this aim, molecular methodologies and cell cultures were combined as a
time-efficient and economical way of studying abalone transcriptomics and cell biology.
Modern Illumina sequencing-by-synthesis technology and subsequent sequence annotation were used
to elucidate differential gene expression between two sibling groups of abalone demonstrating
significant growth variation. Following transcriptome sequencing, genes involved in growth and
metabolism, previously unknown in H. midae, were identified. The expression of selected target genes
involved in growth was subsequently analyzed by quantitative real-time PCR (qPCR).
The feasibility of primary cell cultures for H. midae was furthermore investigated by targeting embryo,
larval and haemolymph tissues for the initiation of primary cell culture. Larval cells and haemocytes
could be successfully maintained in vitro for limited periods. Primary haemocyte cultures demonstrated
to be a suitable in vitro system for studying gene expression and were subsequently used for RNA
extraction and qPCR, to evaluate differential growth induced by bovine insulin and epidermal growth
factor (EGF).
Gene expression was thus quantified in fast and slow growing abalone and in in vitro primary
haemocyte cultures treated with different growth stimulating factors. The results obtained from
transcriptome analysis and qPCR revealed significant differences in gene expression between large and
small abalone, and between treated and untreated haemocyte cell cultures. Throughout in vivo and in
vitro qPCR experiments, the up-regulation of genes involved in the insulin signaling pathway provides
evidence for the involvement of insulin in enhanced growth rate for various H. midae tissues.
Besides the regulation of target genes, valuable knowledge was also gained in terms of reference genes,
during qPCR experimentation. By quantifying the stable expression of two genes (8629, ribosomal
protein S9 and 12621, ornithine decarboxylase) in various tissues and under various conditions, suitable
reference genes, that can also be used in future H. midae qPCR studies, were identified.
By providing evidence at the transcriptional level for the involvement of insulin, insulin-like growth
factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs) in improved growth rate of H.
midae, the relevance of investigating ways to stimulate insulin/IGF release in aquaculture species was
again emphasized. As nutritional administration remains the most probable route of introducing agents that can stimulate the release of insulin-related peptides, continuous endeavours to stimulate abalone
growth through a nutritional approach is encouraged.
This is the first time next generation sequencing is used towards the large scale transcriptome
sequencing of any haliotid species and also the first time a comprehensive investigation is launched
towards the establishment of primary cell cultures for H. midae. A considerable amount of sequence
data was furthermore annotated for the first time in H. midae. The results obtained here provide a
foundation for future genetic studies exploring ways to optimise the commercial production of H.
midae. === AFRIKAANSE OPSOMMING: Die stadige groeitempo van Haliotis midae belemmer die optimale kommersiele produksie van hierdie
mees winsgewende Suid-Afrikaanse akwakultuur spesie. Tot op hede is geen omvattende poging
aangewend om gene verwant aan groeivariasie in H. midae te identifiseer nie. Die doel van hierdie
studie was dus om groeivariasie in H. midae te ondersoek en om spesifieke groei-gekoppelde gene te
identifiseer en hul uitdrukking te kwantifiseer. Ter bereiking van hierdie doel is molekulêre metodes en
selkulture gekombineer as 'n en tydsbesparende en ekonomiese manier om perlemoen transkriptomika en
selbiologie te bestudeer.
Moderne Illumina volgordebepaling-deur-sintese tegnologie en daaropvolgende annotasie is gebruik
om verskille in geenuitdrukking tussen naby-verwante groepe perlemoen, wat noemenswaardige
groeivariasie vertoon, toe te lig. Na afloop van die transkriptoom volgordebepaling is gene betrokke by
groei en metabolisme, vantevore onbekend in H. midae, geïdentifiseer. Die uitdrukking van uitgesoekte
teikengene betrokke by groei is vervolgens ge-analiseer deur kwantitatiewe "real-time PCR" (qPCR). die lewensvatbaarheid van 'n primêre selkulture vir H. midae is ook ondersoek deur embrio, larwe en
hemolimf weefsels te teiken vir die daarstelling van primêre selkulture. Larweselle en hemosiete kon in
vitro suksesvol onderhou word vir beperkte periodes. Primêre hemosietkulture het geblyk 'n gepaste in
vitro sisteem te wees om geenuitdrukking te bestudeer en dit is vervolgens gebruik vir RNS ekstraksie
en qPCR, om differensiële groei, geïnduseer deur insulien en epidermale groeifaktor (EGF), te evalueer.
Geenuitdrukking is dus gekwantifiseer in vinnig- en stadiggroeiende perlemoen en in in vitro primêre
hemosiet selkulture wat behandel is met verskillende groei stimulante. Die resultate wat verkry is van
transkriptoomanalise en qPCR het noemenswaardige verskille in geenuitdrukking tussen groot en klein
perlemoen, en tussen behandelde en onbehandelde hemosiet selkulture uitgelig. Die op-regulering van
gene betrokke by die insulien sein-padweg, tydens in vivo en in vitro qPCR eksperimente, bied getuienis
vir die betrokkenheid van insulien in die verhoogde groeitempo van verskeie H. midae weefsels.
Benewens die regulering van teikengene is waardevolle kennis ook ingewin in terme van
verwysingsgene tydens qPCR eksperimentering. Deur die stabiele uitdrukking van twee gene (8629,
ribosomale proteien S9 en 12621, ornitien dekarboksilase) te kwantifiseer in verskeie weefsels en onder
verskeie kondisies is gepaste verwysingsgene, wat ook in toekomstige H. midae qPCR eksperimente
aangewend kan word, geïdentifiseer.
Deur getuienis vir die betrokkenheid van insulien, insuliensoortige groeifaktor en insuliensoortige
groeifaktor-bindingsproteïene by verbeterde groei van H. midae op transkripsievlak te bied, is die
toepaslikheid van bestudering van maniere om insulienvrystelling in akwakultuurspesies te stimuleer, beklemtoon. Aangesien voeding die mees waarskynlike roete is om middele wat insuliensoortige
peptiedvrystelling stimuleer daar te stel, word vogehoue pogings om perlemoengroei deur die regte
voeding te stimuleer, aangemoedig.
Hierdie is die eerste studie wat volgende generasie volgordebepaling (“next generation sequencing”)
gebruik vir die grootskaalse transkriptoom volgordebepaling van enige haliotied spesie. Dit is ook die
eerste keer dat ‘n omvattende ondersoek geloods word na die daarstelling van primêre selkulture vir H.midae. ‘n Aansienlike hoeveelheid volgorde data is ook vir die eerste keer geannoteer in H. midae. Die
resultate wat hier verkry is bied ‘n basis vir toekomstige genetiese studies wat maniere ondersoek om
die kommersiële produksie van perlemoen te optimiseer.
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