- Title
- Influence of metal exposure history on metal tolerance in the Sydney rock oyster (Saccostrea glomerata)
- Creator
- Yingprasertchai, Thanvapon Senee
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2016
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Tolerance to heavy metal pollution has been described in many living organisms exposed to heavy metals at sublethal levels. Organisms living in metal contaminated sites have to develop physiological processes to respond to metal exposure to ensure survival and reproduction. The organisms may respond to this challenge through physiological acclimation or adaptation (the selection of resistant genotypes). This thesis was focused on the influence of prior metal exposure history on metal tolerance in Sydney rock oyster, Saccostrea glomerata in NSW, Australia. The thesis explored both physiological acclimation and possible adaptation to metal stress. To determine the level of metal contamination among estuaries in NSW and indicate the most relevant metals for further experimental studies, multivariate statistical techniques such as principle component analysis (PCA) and factor analysis (FA), and ecological risk indices including contamination factor (Cf), degree of contamination (Cd) and pollution loading index (PLI) were employed. Using accumulated metals in tissues of the oysters growing wild and farmed in NSW, these analyses identified locations both low and elevated in metals and identified metals most responsible for driving differences among locations.. The results indicated that most of polluted estuaries were located in the Central region of NSW, having impacts from large urban and industrial conurbation, while the unpolluted estuaries were located in the Northern / Southern region within the major regional waterways with low urban activity. The most elevated metals among NSW estuaries were cadmium, copper, lead and zinc. To investigate possible adaptation, the influence of metal exposure history on metal tolerance in the Sydney rock oyster (Saccostrea glomerata) offspring was investigated. The oysters were sampled from ten NSW estuaries such as Clyde River, Hastings River, Hunter River, Manning River, Nambucca, North Haven, Port Stephens, Port Kembla, Swansea Channels, and Wallis Lake which have a gradient in metal exposure history based on the results of multivariate analysis (PCA/FA) and ecological indices (Cf, Cd and PLI) of metals accumulated to adult oysters. Embryos were prepared under laboratory conditions by fertilizing sperm from ten males and oocytes from ten females within estuary using a strip spawning technique. The embryos were then exposed to sublethal concentrations of 2.5-40 μg/l, copper and 7.5-120 μg/l zinc for 48 h. The median effective concentration (EC50) was determined as the metal concentration that caused 50% abnormal D-veliger larvae development.. The EC50 values for copper were not significantly different among estuaries. Interestingly, a strong positive correlation between EC50 and tissue metal concentration was found for zinc (R² = 0.835, p<0.000), but not for copper. Locations with higher environmental Zn exposures had more tolerant offspring. Such a finding suggests adaptation to Zn exposure (the selection of resistant genotypes) or a physiological acclimation effect mediated via maternal transfer. The full-length genomic sequence of S. glomerata, metallothionein (sgMT) was cloned and characterized. The sgMT sequence was composed of two metal responsive elements (MRE), one TATA box, one activator protein-1 (AP-1), three coding exons (28, 117, 80 bp), and two introns (115, 393 bp). The coding exons encoding a protein of 74 amino acids, with 9 cysteine motifs (Cys-X-Cys), cysteine-rich (28%), high lysine content (13.5%) and no aromatic amino acid residues. The phylogenetic analysis of sgMT protein sequence revealed that the sgMT was classified into the MT isoform I. Sequencing of full length of MT mRNA and MT genomic DNA allowed the identification of an intron-exon boundary and the development of the quantitative real time polymerase chain reaction (qRT-PCR). The qRT-PCR revealed that tissue-specific MT gene expression in S. glomerata was expressed highest in the digestive gland; significantly higher than gills, mantle, adductor muscle and gonad. The digestive gland was selected as a target tissue for a further examination of S. glomerata MT gene expression. Physiological acclimation to metals was also investigated by comparing adult oysters with a past history of metal exposure to oysters with no past history of metal exposure (both from the same gene pool) and examining their responses to subsequent metal challenges. Specifically, the experiment involved examining the influence of prior metal exposure history in wild Sydney rock oyster (Saccostrea glomerata) on metal accumulation, MT mRNA expression, glutathione peroxidase (GPx) and superoxide dismutase (SOD) upon subsequent cadmium, copper and zinc exposure. Oysters were sampled from seven locations in Hunter River such as Fullerton Cove, Kooragang Dykes, Windmill, South Arm Bridge, Fern bay, Fullerton St Stockton and Stockton Bridge. The results of multivariate analysis (PCA/FA) and ecological indices (Cf and Cd) of metal accumulation in the oyster tissue from seven locations elucidated that Windmill was the most contaminated with metals, while Fern Bay was the lowest. Copper and zinc were the most elevated metals with 3.71 and 2.51 times higher than NSW background, thus these metals were selected as metal candidates for metal exposure experiment. Cadmium was included as a positive control. Windmill(high past metal exposure history) and Fern Bay( negligible past metal exposure history)oysters were exposed to 20 and 200 μg/l of cadmium, 50 and 500 μg/l of copper and 200 and 2000 μg/l of zinc for 14 days. After exposure, MT mRNA expression, GPx and SOD activities were measured in digestive glands. The two-way ANOVA results revealed that Windmill oysters activated MT mRNA expression significantly higher than Fern bay oysters after exposure to 200 μg/l Cd, F(2, 22) = 10.35, p = 0.0007 and 2000 μg/l Zn, F(2, 21) = 24.28, p < 0.0001. The GPx activity was not significantly different between those oysters. The SOD activity showed significant interaction effects for copper with F(2, 21) = 10.91, p = 0.0006, indicating that Windmill oysters produced SOD activity significantly higher than Fern bay oysters after 50 μg/l Cu exposure. Significant interaction was also found for zinc with F(2, 24) = 7.27, p = 0.0034, where Fern bay oysters produced SOD higher than Windmill oysters after 200 μg/l Zn exposure, while Windmill oysters produced SOD higher than Fern bay oysters after 2000 μg/l Zn exposure. Thus prior exposure to metals can result in an upregulated compensatory response upon subsequent exposure to metals indicative of acclimation. Both acclimation, and potentially adaptation, are mechanisms responsible for the observed tolerance to metals in the Sydney Rock Oyster.
- Subject
- metal tolerance; metallothionein; <i>Saccrotrea glomerata</i>; acclimation; adaptation; oxidative stress; glutathione peroxidase; superoxide dismutase
- Identifier
- http://hdl.handle.net/1959.13/1312288
- Identifier
- uon:22365
- Rights
- Copyright 2016 Thanvapon Senee Yingprasertchai
- Language
- eng
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