Growth-related gene expression in haliotis midae

[摘要] ENGLISH ABSTRACT: The slow growth rate of Haliotis midae impedes the optimal commercial production of this mostprofitable South African aquaculture species. To date, no comprehensive effort has been made toidentify genes associated with growth variation in farmed H. midae. The aim of this study was thereforeto investigate growth variation in H. midae and to identify and quantify the expression of selectedgrowth-related genes. Towards this aim, molecular methodologies and cell cultures were combined as atime-efficient and economical way of studying abalone transcriptomics and cell biology.Modern Illumina sequencing-by-synthesis technology and subsequent sequence annotation were usedto elucidate differential gene expression between two sibling groups of abalone demonstratingsignificant growth variation. Following transcriptome sequencing, genes involved in growth andmetabolism, previously unknown in H. midae, were identified. The expression of selected target genesinvolved 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 haemocytescould be successfully maintained in vitro for limited periods. Primary haemocyte cultures demonstratedto be a suitable in vitro system for studying gene expression and were subsequently used for RNAextraction and qPCR, to evaluate differential growth induced by bovine insulin and epidermal growthfactor (EGF).Gene expression was thus quantified in fast and slow growing abalone and in in vitro primaryhaemocyte cultures treated with different growth stimulating factors. The results obtained fromtranscriptome analysis and qPCR revealed significant differences in gene expression between large andsmall abalone, and between treated and untreated haemocyte cell cultures. Throughout in vivo and invitro qPCR experiments, the up-regulation of genes involved in the insulin signaling pathway providesevidence 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, ribosomalprotein S9 and 12621, ornithine decarboxylase) in various tissues and under various conditions, suitablereference 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 growthfactors (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 wasagain 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 abalonegrowth through a nutritional approach is encouraged.This is the first time next generation sequencing is used towards the large scale transcriptomesequencing of any haliotid species and also the first time a comprehensive investigation is launchedtowards the establishment of primary cell cultures for H. midae. A considerable amount of sequencedata was furthermore annotated for the first time in H. midae. The results obtained here provide afoundation for future genetic studies exploring ways to optimise the commercial production of H.midae.

[发布机构] Stellenbosch University