Letteratura scientifica selezionata sul tema "Saccostrea glomerata"

Larvae are a critical dispersal stage of marine invertebrates, and their survival depends on nutrition and energetics. This study compared the size, survival, metabolic rate and egg and larval lipid class profiles of larvae of the endemic Sydney rock oyster Saccostrea glomerata and the invasive Pacific oyster Magallana gigas through a period of starvation for 5 and 9 d after fertilisation. Starved larvae grew without food until 5 d of age, at which point they stopped developing, but resumed growth when fed. Egg lipids profiles comprised 78.1 and 74.5% triacylglycerol for M. gigas and S. glomerata respectively. When fed, larvae of M. gigas were significantly larger in size and had faster growth and similar survival compared to S. glomerata. When starved, larvae of M. gigas and S. glomerata grew at similar rates, and there was a trend for lower survival of M. gigas. Larval endogenous lipid reserves were deleted in the first 24 h. Larvae of M. gigas had more total lipids and comparatively more diacylglycerols, monoacylglycerols, phospholipids and cholesterol, whereas S. glomerata had more diacylglycerols and produced sterol esters. Starvation altered the patterns of lipid assimilation, and metabolic rates of larvae of M. gigas and S. glomerata differed over time. When starved, S. glomerata larvae had greater capacity to cope with starvation compared to M. gigas, perhaps due to an evolutionary history in oligotrophic estuaries. As the climate rapidly changes in this global climate-change hotspot, S. glomerata is likely to be negatively affected.

Marine diseases represent a significant threat to wild organisms and the ecosystem services they support, yet studies often consider only disease impacts to aquaculture. In eastern Australia, the Sydney rock oyster (Saccostrea glomerata) aquaculture industry is increasingly affected by outbreaks of QX disease caused by parasitic Marteilia sydneyi. The present study considered impacts of M. sydneyi infection on the structure of wild-oyster populations that are dominated by S. glomerata, but that may also include the non-native Pacific oyster, Crassostrea gigas. In the Hawkesbury River Estuary, where cultured S. glomerata has experienced up to 98% QX-induced mortality, we found that disease prevalence was comparatively low among wild S. glomerata, peaking at 14%, and annual infections did not cause seasonal patterns of mortality. Furthermore, C. gigas, a competitor of S. glomerata that is not susceptible to QX disease, was not consistently more abundant at sites with than without the parasite. Overall, our results indicated that relative to cultured counterparts, wild S. glomerata in the Hawkesbury River Estuary is minimally affected by QX disease. Nevertheless, our study showed that diseases of aquaculture stocks have the capacity to infect wild populations, and that longer-term assessment of wild populations at risk is essential.

Estuarine acidification, caused by disturbance of acid sulfate soils (ASS), is a recurrent problem in eastern Australia. Affected waters are characterised by low pH and elevated concentrations of metals, principally aluminium and iron. The effects of acid and elevated metal concentrations associated with ASS, on adult Sydney rock oysters, have not been previously investigated. This study tested links between ASS-affected drainage, subsequent estuarine acidification and Sydney rock oyster production problems on the Hastings and Manning Rivers, mid north coast New South Wales. The primary objective of this thesis was to establish if estuarine acidification causes mortality and slow growth in individual Sydney rock oysters by exposing oysters to low pH, iron and aluminium using field and laboratory experiments. Water quality data showed that estuarine acidification was spatially extensive in the Hastings and Manning Rivers following heavy rainfall and was due to mineral acids originating from drained or excavated ASS. Estuarine acidification regularly affected areas used for Sydney rock oyster production following heavy rainfall. Field experiments showed that Sydney rock oyster mortality rates were significantly higher at sites exposed to ASS-affected waters compared to locations that were isolated from ASS-affected waters. Oyster mortality increased with the time of exposure and smaller oysters (mean weight = 5 g) experienced significantly higher mortality relative to larger oysters (mean weight = 29 g). This was caused by acid-induced shell degradation resulting in perforation of the smaller oysters??? under-developed shells. Additionally, Sydney rock oyster growth rates were dramatically reduced at sites exposed to ASS-affected waters and the overall mean condition index of oysters at ASS-affected field sites was significantly lower than the overall mean condition index of oysters at non-impacted sites. Findings from laboratory experiments showed that ASS-affected water alters oyster valve movements and significantly reduces oyster feeding rates at pH 5.5. Acidic treatments (pH 5.1) containing 7.64 mg L-1 of aluminium or ASS-affected water caused changes in the mantle and gill soft tissues following short-term exposure. Degenerative effects described in oysters in this study were also due to iron contained in ASS-affected waters. Iron precipitates accumulated on the shell, gills and mantle and were observed in the stomach, intestine, digestive tubules and rectum. This study concluded that Sydney rock oysters are unable to tolerate acidic conditions caused by ASS outflows and cannot be viably cultivated in acid-prone areas of the estuary.

Genetic variation is the resource animal breeders exploit in stock improvement programs. Both the process of selection and husbandry practices employed in aquaculture will erode genetic variation levels overtime, hence the critical resource can be lost and this may compromise future genetic gains in breeding programs. The amount of genetic variation in five lines of Sydney Rock Oyster (SRO) that had been selected for QX (Queensland unknown) disease resistance were examined and compared with that in a wild reference population using seven specific SRO microsatellite loci. The five selected lines had significantly lower levels of genetic diversity than did the wild reference population with allelic diversity declining approximately 80%, but impacts on heterozygosity per locus were less severe. Significant deficiencies in heterozygotes were detected at six of the seven loci in both mass selected lines and the wild reference population. Against this trend however, a significant excess of heterozygotes was recorded at three loci Sgo9, Sgo14 and Sgo21 in three QX disease resistant lines (#2, #5 and #13). All populations were significantly genetic differentiated from each other based on pairwise FST values. A neighbour joining tree based on DA genetic distances showed a clear separation between all culture and wild populations. Results of this study show clearly, that the impacts of the stock improvement program for SRO has significantly eroded natural levels of genetic variation in the culture lines. This could compromise long-term genetic gains and affect sustainability of the SRO breeding program over the long-term.

There has been a continuous decline in both the production and general performance of the SRO in NSW estuaries over the past three decades. The relationship of this decline to both environmental and oyster-density related factors are assessed in this thesis. This question has been examined at different scales: a large scale that compares two different estuaries (Clyde and Shoalhaven Rivers, southern NSW); a regional scale that encompasses variations within an estuary and, at a lease scale that examines processes pertaining to individual or small groups of oysters. Levels of inorganic nutrients were in general very low potentially limiting primary production. The limiting nutrient was nitrogen or phosphorus depending on whether long term conditions were dry or wet, respectively. Only during rain events, through the input of terrestrial material, were conditions favourable for fast rates of primary production. Carbon and nitrogen isotope analysis has demonstrated that both external material and local resuspension of the benthos constitute a major proportion of the SRO diet. The uptake of the various food sources also varied considerably depending on local environmental conditions. Increases in SRO growth were strongly correlated to increases in temperature with a low temperature cut-off at ~13°C. Growth also appeared to reduce considerably when salinities lower than ~15ppt persisted for the order of a month. These factors may alter growth through changes in filtration rates. These processes were modelled in a coupled hydrodynamic-NPO (Nitrogen-Phytoplankton-Oyster) model of the Clyde River. This demonstrated that primary production was more affected by estuarine dynamics and nutrient concentrations than oyster uptake. At the current levels of oyster densities, primary production by itself could not account for the observed oyster growth, however growth became realistic with observed levels of POC added to the model. A set of environmental indices were used to complement the model and to assess the sustainability of the culture system. The combined indices indicated that while the ecological carrying capacity of the Clyde was exceeded the production capacity at an estuarine scale was not. On the lease scale, density experiments showed that while growth was not reduced as a result of current stocking densities, the condition index was significantly affected.

Research Doctorate - Doctor of Philosophy (PhD)
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 (C_f), degree of contamination (C_d) 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 (C_f, C_d 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 (C_f and C_d) 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.

Research Doctorate - Doctor of Philosophy (PhD)
Insulin-like growth factors (IGFs) play crucial roles in regulating growth, development and metabolism in animals. IGFs are complexed with specific binding proteins known as IGF binding proteins (IGFBPs), which modulate, either stimulate or inhibit, the action of IGFs. Most of the current literature on oyster biology focuses on reproductive cycle and growth patterns as well as their relationship with their ambient environmental conditions. Compared with vertebrates, little is known about the biology of IGFBPs and the molecular mechanisms involved in the regulation of growth in invertebrates, particularly molluscs. To fill this knowledge gap, both the cDNA and genomic DNA sequences of the IGFBP-5 and IGFBP-7 homologues were cloned from the Sydney rock oyster (SRO), Saccostrea glomerata. The spatial and temporal mRNA expression patterns, single nucleotide polymorphisms (SNPs) and their consequent haplotypes of these two genes as well as their transcriptional responses to fasting and hypoxia were investigated to decipher their potential roles in the regulation of the SRO growth. The full-length cDNA and gDNA sequences of the S. glomerata IGFBP-5 and IGFBP-7 genes (designated sgIGFBP-5 and sgIGFBP-7, respectively) were cloned and characterised for the first time. For sgIGFBP-5, the ORF consists of 357 bp, encoding a polypeptide of 118 amino acid residues. The deduced amino acid sequence of sgIGFBP-5 contained conserved N- and C-terminal regions, multiple cysteine residues and two conserved motifs (GCGCCXXC and CWCV). There are multiple putative regulatory DNA elements in the promoter region of sgIGBFP-5 which might interact with specific transcription factors to drive gene transcription, including an ERE half site, a SP1 site, an AP-1 site and a HRE. The genomic DNA of sgIGBFP-5 consists of 3 exons and 2 introns. For sgIGFBP-7, its ORF comprises of 780 bp, encoding a polypeptide of 259 amino acid residues. The sgIGFBP-7 protein consists of three conserved domains, including the IGFBP N-terminal domain, the Kazal-type serine protease inhibitor domain and the immunoglobin (Ig) domain, as well as one conserved motif “xCGCCxxC”. The promoter region of sgIGFBP-7 comprises of multiple putative regulatory DNA elements, including an ERE half site, an EGR1 binding site, a NF-κB cells binding site and a HRE core sequence. Phylogenetic analysis showed that the deduced proteins of sgIGFBP-5 and sgIGFBP-7 are clustered with their corresponding homologues from other molluscan species, supporting the proper nomenclature of the SRO genes. All of the observations above suggest that sgIGBFP-5 and sgIGBFP-7 is authentic IGFBP homologues and their genomic and deduced protein structures are conserved evolutionarily. Expression of sgIGFBP-5 and sgIGFBP-7 mRNA was detected in all the tested tissues in both juvenile and adult oysters. Notably, contrasting differential expression profiles of sgIGFBP-5 and sgIGFBP-7 mRNAs between the adult slow-growing and fast-growing oysters were observed in certain tissue types, implying that IGFBPs may play potential roles in regulating the growth of SRO. DNA sequencing of 100 oysters from each of the slow and fast-growing lines revealed the presence of 183 and 76 SNPs in the genomic regions of sgIGFBP-5 and sgIGFBP-7, respectively. Among these, 7 SNPs in sgIGFBP-5 and 16 SNPs in sgIGFBP-7 are significantly different in genotype/allele frequencies between the two oyster lines. The association of the SNPs identified in sgIGFBP-5 and sgIGFBP-7 with growth traits was examined among the oyster individuals grouped according to their genotypes constituted by each SNP. For sgIGFBP-5, significant differences were found in shell length among the genotypes constituted by six SNPs and in shell height among the genotypes constituted by all seven SNPs. For sgIGBFP-7, significant differences in shell length, shell height and whole oyster weight were found among the genotypes constituted by the majority of the identified SNPs. The association of sgIGFBP-5 and sgIGFBP-7 haplotypes with growth traits was also investigated. Statistically significant differences among the haplotypes were observed with respect to shell length and shell height for sgIGFBP-5 while no significant difference among haplotypes with respect to any of the tested growth traits for sgIGFBP-7 was found. These results suggest that the SNPs/haplotypes in sgIGFBP-5 and sgIGFBP-7 may have a significant association with the growth traits in S. glomerata and hence they may provide the basis for developing marker-assisted selection strategies to improve the oyster’s growth performance in the future. An in silico protein-protein interaction analysis was performed to identify the strengths of the potential binding between the deduced protein of each nonsynonymous haplotype groups and their potential ligands. The results revealed that the orders of binding strength of the tested ligands with each of the sgIGFBP-5 and sgIGFBP-7 haplotype groups were different. This finding suggests that sgIGFBP-5 and sgIGFBP-7 may have their own binding preferences for different cellular ligands, implying that the non-synonymous SNPs presented in the haplotype groups may give rise to critical changes in the 3D structure of the encoded IGFBP proteins, which in turn alter IGFBP-ligand interaction. A short-term starvation experiment and investigation of the regulatory effect of hypoxia on mRNA expression of sgIGFBP-5 and sgIGFBP-7 were conducted. For the fasting experiment, the sgIGFBP-5 and sgIGFBP-7 expression levels in both digestive gland and mantle tissue in fasting oysters increased compared to controls at the time points of Days 3, 5 and 7. This lead to the postulation that catabolic activity was increased and IGFBPs in the starved oysters are released into the circulation to bind to IGFs, thereby restricting IGFs from binding to their cognate receptors and hence slow down the oysters’ growth. The study also pointed out that re-feeding (2 days) of fasting oysters restored the basal expression of the IGFBP mRNA. This may suggest that the downregulation of these genes could in turn release the availability of IGFs for IGF-receptor binding or growth. However, this was the short-term starvation experiment, and the changes in growth traits of S. glomerata were not measured. Thus, it remains premature to conclude the roles of the sgIGFBPs in growth regulation. For the hypoxic experiment, no significant change in HIF-1 and IGFBP mRNA expression were detected in any of the tested oyster tissues after 5 days of air exposure. These observations do not agree with a hypothesis that HIF-1 expression is increased under hypoxic conditions and the increased HIF-1 availability can in turn activate IGFBP gene transcription via binding to the HRE(s) located in the IGFBP gene promoter. Overall, the findings from this research provide information on the structures and potential functions of IGFBPs in relation to growth and stress effects. In the future, from the SNPs and/or haplotypes in sgIGFBP-5 and sgIGFBP-7, identification of growth associated genetic markers will contribute to improving the genetic stock, productivity, sustainability and profitability of the SRO (and potentially other molluscan species).

There has been a continuous decline in both the production and general performance of the SRO in NSW estuaries over the past three decades. The relationship of this decline to both environmental and oyster-density related factors are assessed in this thesis. This question has been examined at different scales: a large scale that compares two different estuaries (Clyde and Shoalhaven Rivers, southern NSW); a regional scale that encompasses variations within an estuary and, at a lease scale that examines processes pertaining to individual or small groups of oysters. ... A set of environmental indices were used to complement the model and to assess the sustainability of the culture system. The combined indices indicated that while the ecological carrying capacity of the Clyde was exceeded the production capacity at an estuarine scale was not. On the lease scale, density experiments showed that while growth was not reduced as a result of current stocking densities, the condition index was significantly affected.

Research Doctorate - Doctor of Philosophy (PhD)
Environmental estrogens are known to interfere with normal development and reproduction in a wide range of marine invertebrates. Previous studies have developed an oyster biomonitor to indicate the presence of estrogenic compounds in marine environments through exploiting the induction of the egg yolk protein precursor vitellogenin (Vtg). Despite this advance, the mechanism through which estrogens exert their action on Vtg gene expression, in particular the functional role of the estrogen receptor (ER), is currently unknown. In an attempt to fill this knowledge gap, the present research aims to isolate the genes encoding Vtg (sgVtg) and ER (sgER) from the Sydney rock oyster Saccostrea glomerata and investigate different potential mechanisms contributing to estrogen mediated induction of sgVtg. Our results indicated that the deduced protein of sgVtg is substantially longer than those of the other oyster Vtgs reported so far and contains all the conserved domains as found in other marine molluscs. The sgVtg promoter contains multiple putative half-EREs which are closely spaced, implying that they may function as an estrogen response unit (ERU) to interact with ER. In line with the potential involvement of ER in sgVtg regulation, the induction of sgVtg mRNA expression in ovarian explants was shown to be abolished by the ER antagonist ICI 182, 780. Considering that the vertebrate-like ER so far reported in molluscs lacks estrogen-binding ability, this finding supports the requirement of a novel estrogen-binding receptor for gene activation. The sgER cDNA is predicted to encode a 477-amino acid protein, which contains a DNA binding domain (DBD) and a ligand binding domain (LBD) conserved among vertebrate and invertebrate ERs. Comparison of the sgER LBD sequence with those of other ligand-dependent ERs indicated that the sgER LBD is degenerate at several conserved residues critical for ligand binding and receptor activation. Its inability to bind estrogens was then confirmed by a ligand binding assay using fluorescent-labelled E2 and purified sgER protein. The 5′-flanking region of sgER contains three putative ½EREs and several other putative elements for ER-interacting transcription factors, suggesting potential autoregulation of sgER expression. sgER mRNA is ubiquitously expressed in various tissues, with the highest expression level observed in the ovary where sgVtg is highly expressed. Functional analyses, including luciferase gene reporter assays, point mutation of ½ EREs and an electrophoretic mobility shift assay (EMSA), confirmed that sgER binds and activates the sgVtg promoter through ½EREs. In addition, sgER mRNA was significantly upregulated following in vitro and in vivo exposure to E2 and the enhancing effect of E2 on sgER expression was abolished by co-treatment with the specific ER antagonist ICI 182, 780 in vitro. These findings support the presence of a novel estrogen-binding receptor in S. glomerata. To elucidate whether estrogens modulate sgVtg and sgER expression at the epigenetic level, we assessed the DNA methylation levels of a 5’ intragenic CpG island in sgVtg and a promoter CpG island in sgER in ovaries after E2 exposure in vivo. Bisulfite sequencing revealed that both of these CpG islands are hypomethylated in both control and E2-treated oysters. Neither significant differential DNA methylation nor correlation between DNA methylation and mRNA levels was observed for either sgVtg or sgER. Overall, the findings from this research provides new molecular insights into how environmental estrogens regulate Vtg expression in marine molluscs and lays the foundation for further research into the mechanism of action of estrogenic compounds on molluscan vitellogenesis.