Molecular Genetics of Immunity in the Sydney Rock Oyster (Saccostrea glomerata)

Timothy Green (2009). Molecular Genetics of Immunity in the Sydney Rock Oyster (Saccostrea glomerata) PhD Thesis, School of Biological Sciences, The University of Queensland.

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Author Timothy Green
Thesis Title Molecular Genetics of Immunity in the Sydney Rock Oyster (Saccostrea glomerata)
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2009-10
Thesis type PhD Thesis
Supervisor Andrew C. Barnes
Robert Adlard
Total pages 143
Total colour pages 3
Total black and white pages 140
Subjects 06 Biological Sciences
Abstract/Summary Mass mortalities of farmed Sydney rock oyster, Saccostrea glomerata, have been observed in Australia since the 1970s and are attributed to the paramyxean protozoan parasite, Marteilia sydneyi (etiological agent of QX disease). This opportunistic parasite infects S. glomerata when the oyster’s immune system has been compromised due to one or more unknown transient environmental stressor. Management and prevention of the disease is seriously compromised as neither the risk factors for the disease, nor the complete life cycle of M. sydneyi are currently understood. The future of the rock oyster industry relies on the development of QX-resistant oysters. Selection of S. glomerata for resistance to QX disease over several generations has revealed that QX-resistance is heritable and likely to be controlled by multiple genes. The main focuses of this thesis was to improve our knowledge of the genes involved in immunity of S. glomerata to disease. The transcriptome response of hemocytes isolated from S. glomerata selected over four generations for resistance to QX disease (QXR4) was compared to non-selected control oysters (W-type) using suppression, subtractive hybridization (SSH) and quantitative real-time PCR (qRT-PCR). Our data supported that differences in gene expression due to selection was largely attributable to constitutive differences in transcriptional rate. The basal-expression of an extracellular superoxide dismutase (EcSOD) and small heat shock protein (sHsP) was 3.18- and 2.05-fold higher in QXR4 oysters, respectively (p < 0.5). The basal-expression of peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine (IK) was 2.75- and 1.5-fold lower in QXR4 oysters, respectively (p < 0.5). Expression of EcSOD, Prx6 and IK was measured in S. glomerata in response to injection with a range of pathogen associated molecular patterns (PAMPs) to replicate microbial invasion as QX disease cannot be replicated in the laboratory due to the complex life cycle of M. sydneyi. The expression of IK was induced 2.2-fold in S. glomerata in response to injection with double stranded RNA when compared to control oysters (p < 0.05). However, changes in the expression of EcSOD and Prx6 could not be induced with any of the tested PAMPs (p > 0.5), suggesting the difference in basal-expression of these two genes between QXR4 and W-type oysters would be maintained during infection with M. sydneyi. It was concluded that QXR4 oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H2O2) faster due to the elevated levels of EcSOD and that the H2O2 would reach higher concentrations due to the reduced levels of Prx6 to detoxify it. Attempts were made to understand why the basal expression of EcSOD and Prx6 is different between QXR4 and W-type oysters. Identification of DNA variants within the promoter regions of these genes would provide greater insight into the gene network(s) involved in disease resistance and possibly lead to identification of QX-resistant markers that can be applied to the Sydney rock oyster breeding program. The differential expression of EcSOD and Prx6 is likely to originate from either modifications in transcriptional rate or mRNA stability as the percentage of hemocytes that express these two genes was shown to be equal between QXR4 and W-type oysters using in-situ hybridization (p > 0.5). Attempts to amplify and assemble the full-length EcSOD gene from S. glomerata were unsuccessful. The full Prx6 gene was amplified and the frequency of polymorphisms that affected mRNA stability and transcriptional rate were determine between QX-resistant and –susceptible S. glomerata (N = 15). Notably, the frequency of a single nucleotide polymorphism (SNP) in the promoter region (-240A>G) affected the binding of a heat shock factor. The genotypic frequency of -240G/G was 0.400 in resistant oysters compared to 0.067 in susceptible oysters (p = 0.059). Further validation of this SNP is now required using a larger data set. Monitoring survival and histological observations of S. glomerata over the 2006/2007 and 2007/2008 QX disease risk period in the Pimpama River, SE Queensland provided further support to anecdotal evidence that mortality of S. glomerata occurs after heavy summer rainfall in SE Queensland. It is presumed that heavy rainfall causes the immune system of S. glomerata to become compromised, presumably by a reduction in salinity and/or estuarine acidification from a rising water table leading to leaching of acid sulfate soils. Laboratory trials revealed a drop in salinity from 35 ppt to 15 ppt affected immune gene expression and inhibition of the Prx6 gene could still be detected five days after oysters were returned to normal seawater. Acid sulfate soil leachate had no effect on the expression of immune genes or immunological parameters tested. This result provides further support to existing evidence that reduced salinity causes the immune system of S. glomerata to be compromised, possibly resulting in higher mortality of S. glomerata when subsequently challenged with M. sydneyi. Examination of S. glomerata during the QX disease risk period revealed oysters were often infected with other parasites. The microsporidian parasite, Steinhausia sp. was frequently observed infecting the gonad tissue of female S. glomerata in histological sections. This infection resulted in hemocytic infiltration and re-absorption of gonad tissue, possibly resulting in reduced growth rates, condition index and marketablity. The frequency of this parasite in oyster samples taken from Moreton Bay, SE Queensland, suggests this parasite could be an emerging problem for oyster farmers in SE Queensland. Differences in the bacterial community within the digestive gland of S. glomerata infected and un-infected with M. sydneyi was observed using non-culture techniques. Healthy oysters had a diverse bacterial community with 23 different operational taxonomic units (OTUs) identified. In contrast, S. glomerata infected with M. sydneyi had only one OTU present in the digestive gland, which was closely related to a Rickettsiales-like prokaryote (RLP) based on phylogenetic analysis of its 16S rDNA sequence. This RLP may be detrimental to its host during concurrent infection with M. sydneyi and warrants further investigation. Overall, this project demonstrated resistance of S. glomerata to M. sydneyi is likely to involve constitutive differences in gene expression. Identification of DNA variants within the promoter regions of differentially expressed genes may provide further insight into gene regulation within oysters and allow identification of DNA markers for selecting QX-resistant brood-stock. Results presented in this thesis support anecdotal evidence that S. glomerata are more susceptible to M. sydneyi following periods of high rainfall as a result of reduced salinity compromising the immune system of S. glomerata. Histological observation of S. glomerata over the QX-disease risk period revealed that oysters were often infected with a range of other parasites. The presence of these parasites may also compromise the immune system of S. glomerata during the QX-disease risk period. The implications of their presence must be factored in to future breeding and research programmes.
Keyword Oyster, Saccostrea, QX disease, Marteilia, Gene Expression, Immunity, Parasitology.
Additional Notes 55, 115, 121

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Created: Thu, 27 May 2010, 12:38:22 EST by Mr Timothy Green on behalf of Library - Information Access Service