Physiological components of growth differences between individual oysters (Crassostrea gigas) and a comparison with Saccostrea commercialis

Pacific oysters (Crassostrea gigas) of identical age from two genetically distinct lines, one fast growing and the other slow growing, were held at three levels of ration and analysed for physiological traits to explain differences in their rates of growth. The data supported three hypotheses; faster growth was associated with faster rates of consumption of food, reduced metabolic rate at maintenance (i.e., at zero growth), and reduced metabolic costs of growth. A comparison with the Sydney rock oyster, Saccostrea commercialis, based on similar experiments on the two species, indicated that faster growth of Pacific oysters depended on similar physiological differences; the mean metabolic costs of growth, however, were similar in the two species. It is suggested that a general model for genetically linked differences in the growth rate of bivalve molluscs will need to include the processes of metabolic control rather than relying solely on an analysis of the individual components of the energetics of growth.     

The physiological basis for faster growth in the Sydney rock oyster, Saccostrea commercialis

Sydney rock oysters were sampled from a mass selection experiment for growth (the "selected" category) and from a control ("not selected") population and held in the laboratory at three ration levels. We evaluated three models to explain faster rates of growth by selected oysters. Selection resulted in oysters feeding at up to twice the rate and with greater metabolic efficiency than controls. A field experiment confirmed that selection leads to faster rates of feeding across a wide range of food concentrations. Selected oysters also grew more efficiently, at a smaller cost of growth (Cg): mean values for Cg were 0.43 J x J(-1) in selected individuals and 0.81 J x J(-1) in the controls. In contrast, oysters in both categories showed similar metabolic rates at maintenance, i.e., at a ration supporting zero growth. There was no evidence that differential energy allocation affected the balance between total metabolic requirements above and below zero net energy balance. By experimenting with selected and control oysters of different sizes and ages, then standardizing the data for size, we found no effects of age on the differences due to selection. Faster-growing oysters feed more rapidly; invest more energy per joule ingested; show a higher net growth efficiency; and are able to allocate less energy per unit of tissue growth, than slower-growing individuals.     

Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata)

Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca²⁺ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (K_m = 4.45 ± 1.46 mM) than for the diphenol, l-DOPA (K_m = 10.27 ± 1.33 mM). Maximum enzyme activity was evident at 37 °C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.     

Effects of water temperature and salinity on elimination of Salmonella charity and Escherichia coli from Sydney rock oysters (Crassostrea commercialis)

The elimination of Salmonella charity and Escherichia coli from the Sydney rock oyster, Crassostrea commercialis, was examined during commercial purification of oysters under different water temperatures and salinities. Both organisms were rapidly eliminated at 18 to 22 degrees C. Purification was effective but slower at 24 to 27 degrees C and incomplete and inconsistent at temperatures below 17 degrees C. The oysters suffered stress and were not effectively purified at water salinities of 15 to 20% but were rapidly purified at 32 to 47% salinity. Winter-harvested and summer-harvested oysters were purified similarly in water at 18 to 22 degrees C and 32 to 36% salinity.     

Sporulation of Marteilioides branchialis n. sp. (Paramyxea) in the Sydney rock oyster, Saccostrea commercialis: an electron microscope study.

The ultrastructure of sporulation of a new parasite, Marteilioides branchialis (Paramyxea), in the Sydney Rock oyster, Saccostrea commercialis, is described. The development is typical of other Paramyxea whereby a stem cell internally cleaves a secondary cell contained within a vacuole. It differs from other species in the phylum in that each secondary cell produces a single spore composed of two concentric cells, one within a vacuole of the other. This type of sporulation represents the simplest of all known Paramyxea. Infection results in focal gill lesions and was observed concurrently with an epizootic of another paramyxean, Marteilia sydneyi.     

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.     

Effects of water temperature and salinity on elimination of Salmonella charity and Escherichia coli from Sydney rock oysters (Crassostrea commercialis).

The elimination of Salmonella charity and Escherichia coli from the Sydney rock oyster, Crassostrea commercialis, was examined during commercial purification of oysters under different water temperatures and salinities. Both organisms were rapidly eliminated at 18 to 22 degrees C. Purification was effective but slower at 24 to 27 degrees C and incomplete and inconsistent at temperatures below 17 degrees C. The oysters suffered stress and were not effectively purified at water salinities of 15 to 20% but were rapidly purified at 32 to 47% salinity. Winter-harvested and summer-harvested oysters were purified similarly in water at 18 to 22 degrees C and 32 to 36% salinity.     

Detection of Minchinia sp., in rock oysters Saccostrea cuccullata (Born, 1778) using DNA probes

Haplosporidian parasites infect various invertebrate hosts including some commercially important shellfish. Haplosporidium nelsoni (along with Perkinsus marinus) has severely affected Eastern oyster production on the eastern seaboard of the United States and flat oyster production in Europe has been severely impacted by Bonamia ostreae. These parasites are also often present at a very low prevalence and there are a variety of morphologically similar species that can be difficult to differentiate during cytological or histological diagnosis hence the need to develop specific tests. Recently, a Minchinia sp. was described affecting rock oysters (Saccostrea cuccullata) in north Western Australia. In this study, two in situ hybridisation (ISH) assays and a PCR assay have been developed and optimised for use in investigating these parasites. The first ISH assay used a 166bp polynucleotide probe while the second used a 30bp oligonucleotide probe. The specificity of each ISH assay was assessed by applying each probe to a variety of haplosporidian (5), a paramyxian (1) or ciliophora (1) parasites. The polynucleotide probe produced strong hybridisation signals against all of the haplosporidian parasites tested (Minchinia sp., Minchinia teredinis, Bonamia roughleyi, H. nelsoni and Haplosporidium costale) while the oligonucleotide probe recognised only the Minchinia sp. Both probes failed to detect the paramyxian (Marteilia sp.) or the Rhynchodid-like ciliate. The PCR assay amplifies a 220bp region and detected Minchinia sp. DNA from 50ng of genomic DNA extracted from the tissues of infected oysters and 10fg of amplified Minchinia sp. DNA. The assay did not react to oysters infected with H. nelsoni or H. costale. The ability of the PCR and oligonucleotide ISH assay to diagnose Minchinia sp. infected oysters was compared to histological examination from a sample of 56 oysters. The PCR assay revealed 26 infections while histological examination detected 14 infections. The oligonucleotide ISH assay detected 29 infections. The oligonucleotide ISH and PCR assays were found to be significantly more sensitive than histology for detecting the parasite.     

Bacterial diversity of the digestive gland of Sydney rock oysters,Saccostrea glomerata infected with the paramyxean parasite,Marteilia sydneyi

Aims: To determine whether the infestation by the protozoan paramyxean parasite, Marteilia sydneyi, changes the bacterial community of the digestive gland of Sydney rock oysters, Saccostrea glomerata. Methods and Results: Six 16S rDNA clone libraries were established from three M. sydneyi‐infected and three un‐infected oysters. Restriction enzyme analysis followed by sequencing representative clones revealed a total of 23 different operational taxonomic units (OTUs) in un‐infected oysters, comprising the major phyla: Firmicutes, Proteobacteria, Cyanobacteria and Spirocheates, where the clone distribution was 44, 36, 7 and 5%, respectively. Close to half of the OTUs are not closely related to any other hitherto determined sequence. In contrast, S. glomerata infected by M. sydneyi had only one OTU present in the digestive gland. Phylogenetic analysis of the 16S rDNA sequence reveals that this dominant OTU, belonging to the α‐Proteobacteria, is closely related to a Rickettsiales‐like prokaryote (RLP). Conclusions: The microbiota of the digestive gland of Sydney rock oysters is changed by infection by M. sydneyi, becoming dominated by a RLP, and generally less diverse. The bacterial community of un‐infected S. glomerata differs from previous studies in that we identified the dominant taxa as Firmicutes and α‐Proteobacteria, rather than heterotrophic γ‐Proteobacteria. Significance and Impact of the Study: This is the first culture‐independent study of the microbiota of the digestive glands of edible oysters to the species level. The commercial viability of the Sydney rock oyster industry in Australia is currently threatened by Queensland Unknown disease and the changes in the bacterial community of S. glomerata corresponding with infection by M. sydneyi sheds further light on the link between parasite infection and mortality in this economically damaging disease.     

In vitro effects of noradrenaline on Sydney rock oyster (Saccostrea glomerata) hemocytes

Our prior work has shown that the catecholamine hormone, noradrenaline, mediates environmental stress responses in Sydney rock oysters, resulting in impaired immunological function. In the current study, we tested the cellular basis of this stress response. Hemocytes were exposed to noradrenaline in vitro before cell morphology and viability were analyzed. Noradrenaline was shown to induce apoptotic markers, including the loss of mitochondrial membrane potential, DNA fragmentation and plasma membrane blebbing. F-actin appeared to play an important role in the changes observed in hemocytes, being concentrated mostly in the plasma membrane blebs of noradrenaline-treated hemocytes. This may explain why hemocyte adhesion and pseudopodia formation were inhibited by noradrenaline. Cellular dysfunction induced by norarenaline mainly affected the hyalinocyte sub-population of hemocytes, whilst the other major cell type, granulocytes, remained unaffected. Given that hyalinocytes are important immunological effectors, the results of this study help to explain why immunosuppression accompanies noradrenaline-mediated stress responses in oysters.     

Morphological and mitochondrial-DNA analysis of the Indo-West Pacific rock oysters (Ostreidae: Saccostrea species)

Species of Saccostrea are common oysters on Indo-Pacific rockyshores. The taxonomy of this genus is unclear, however, becauseof morphological plasticity among its constituent species. Oystersamples with distinct morphotypes were collected from shoresexperiencing different degrees of wave exposure in Japan, Taiwan,China, Vietnam, Singapore and Australia. Intraspecific and interspecificvariations in shell characters have been identified. Only S.kegaki could be readily differentiated from S. cucullata-B,S. cucullata-C and S. mordax by multivariate shell allometricanalyses. Phylogenetic analyses using partial mitochondrial16S DNA sequences of approximately 513 base pairs identifiedtwo clades that correspond to superspecies, i.e. putative S.cucullata and putative S. mordax, comprising nine and two lineagesfrom the Indo-West Pacific, respectively. These are S. cucullata-A,B, C, D, E, F, G, S. kegaki and S. glomerata in one clade, andS. mordax-A and B in the other. Interspecific sequence divergence(Kimura-2-parameter values) between lineages ranged from 1.02%to 11.74%. Both S. cucullata-C and S. kegaki are restrictedto the northern hemisphere, whereas S. glomerata seems to bea temperate species from the southern hemisphere. S. cucullata-Doccurs only in Taiwan. Other species, i.e. S. cucullata-A, B,D, E and F and S. mordax-A and B, have a wide range on rockyshores in the tropical and subtropical Western Pacific and inboth northern and southern hemispheres. (Received 7 January 2005; accepted 4 November 2005)     

Sporulation of Marteilioides branchialis N. Sp. (Paramyxea) in the Sydney Rock Oyster, Saccostrea commercialis: An Electron Microscope Study

ABSTRACT The ultrastructure of sporulation of a new parasite, Marteilioides branchialis (Paramyxea), in the Sydney Rock oyster, Saccostrea commercialis , is described. The development is typical of other Paramyxea whereby a stem cell internally cleaves a secondary cell contained within a vacuole. It differs from other species in the phylum in that each secondary cell produces a single spore composed of two concentric cells, one within a vacuole of the other. This type of sporulation represents the simplest of all known Paramyxea. Infection results in focal gill lesions and was observed concurrently with an epizootic of another paramyxean, Marteilia sydneyi .     

Spore ornamentation of Minchinia occulta n. sp. (Haplosporidia) in rock oysters Saccostrea cuccullata (Born, 1778)

A Minchinia sp. (Haplosporidia: Haplosporidiidae) parasite was identified infecting rock oysters and morphologically described by Hine and Thorne (2002) using light microscopy and transmission electron microscopy (TEM). The parasite was associated with up to 80% mortality in the host species and it is suspected that the parasite would be a major impediment to the development of a tropical rock oyster aquaculture industry in northern Western Australia. However, attempts to identify the parasite following the development of a specific probe for Haplosporidium nelsoni were unsuccessful. The SSU region of the parasite's rRNA gene was later characterized in our laboratory and an in situ hybridization assay for the parasite was developed. This study names the parasite as Minchinia occulta n sp. and morphologically describes the parasite using histology, scanning electron microscopy and transmission electron microscopy. The non-spore stages were unusual in that they consisted primarily of uninucleate stages reminiscent of Bonamia spp. The parasite's spores were ovoid to circular shaped and measured 4.5 microm-5.0 microm x 3.5-4.1 microm in size. The nucleus of the sporoplasm measured 1.5-2.3 microm and was centrally located. The spores were covered in a branching network of microtubule-like structures that may degrade as the spore matures.     

Haplosporidium sp. (Alveolata: Haplosporidia) associated with mortalities among rock oysters Saccostrea cuccullata in north Western Australia

Haplosporidium sp. is described from rock oysters Saccostrea cuccullata Born, 1778 experiencing epizootics on the northwestern coast of Western Australia. All stages were observed as focal infections in the connective tissue of the gills, or as disseminated infections in the mantle and around digestive diverticulae. Haplosporidium sp. occurred between epithelial cells of the gut, in focal lesions in the gills, but not in the epithelium of the digestive diverticulae, and sporulation was confined to the connective tissue. Plasmodia developed into sporonts and sporocysts in a loose syncytium that gave rise to binucleate and uninucleate sporoblasts from which spores developed. Spores were flask-shaped, 5.6-6.7 x 3.3-4.0 microm, with a characteristic operculum, a few filamentous wrappings and rod-like structures in the posterior sporoplasm. Mature spores had a wall comprising inner (90 nm wide), middle (30 nm wide) and outer (130 nm wide) layers, and a surface coat of microtubules giving them a furry appearance. Oysters with empty gonad follicles were most heavily infected, and oyster condition and mortality appeared to be related to degree of infection.     

Genetic structure and effective population size of Sydney rock oysters in eastern Australia

Oyster reef habitats are critical to coastal biodiversity and their decline has prompted restoration efforts in Australia. Knowledge gaps exist regarding the population structure and diversity of key species in these habitats. This may be critical information for the design of effective restoration programs. Sydney rock oysters (Saccostrea glomerata) are the dominant reef-forming bivalve in eastern Australia. Wild populations of S. glomerata have declined due to overharvesting, disease outbreaks, coastal development and reduced water quality. Here, we use genetic markers identified by genome-wide sequencing to investigate the genetic structure and diversity of wild Sydney rock oysters throughout their distribution in eastern Australia. We examine evidence for past population bottlenecks and spatial genetic structure associated with the East Australian Current. Analysis of 3, 400 neutral single-nucleotide polymorphisms (SNPs) revealed a single population, and an overlap with two other Saccostrea sp. at the northernmost boundary of the distribution. We detected signals of asymmetric gene flow consistent with the direction of the East Australian Current, and spatial structure patterns of limited genetic isolation by distance and spatial autocorrelation in the northern region (which experiences stronger effects of the East Australian Current) but not in the southern region of the distribution. We found no evidence of significant recent bottlenecks, with high effective population size throughout the species’ range. This information will provide a baseline against which to assess the impact of restoration projects, and guide strategies for sourcing stock for the enhancement of wild oyster populations. Our results provide a positive outlook for the resilience and adaptive capacity of Sydney rock oysters, and highlight wild populations as valuable resources for aquaculture and restoration initiatives.

     

Physiological costs of reproduction in the Sydney rock oyster<Emphasis Type="Italic"> Saccostrea glomerata</Emphasis>

In this study, triploid Sydney rock oysters Saccostrea glomerata, which do not reproduce and have only limited gonadal development, were used to calculate the cost of producing and maintaining somatic tissues. The consumption of oxygen was measured and converted to units of energy expended. The consumption of oxygen of diploid oysters, in different stages of the reproductive cycle, was also measured. Knowing the costs of producing and maintaining somatic tissues (obtained from the triploid oysters), it was possible to calculate the energy demand of somatic and reproductive tissues of diploid oysters. The focus of this study was to test whether this method would work, to investigate if this method would give results in accordance with modern life-history theory and to test hypotheses about costs of reproduction in oysters. It was found that in diploid oysters, 27% of the consumed oxygen was needed for reproductive processes. It was also found that the costs of production and maintenance of reproductive tissues were on average 84% of those of somatic tissues. Costs for the production and maintenance of somatic tissues decreased over time. Costs for reproduction also decreased, but were dependent on the stage of gonadal development. If the relative mass of gametes in the gonads was large, the costs were relatively small; if the mass was relatively small, the costs were large. Differences between traits of males and females were never significant, suggesting that reproductive effort and costs were similar in males and females. It was estimated that if diploid oysters did not reproduce, they could gain 64% more somatic ash-free dry mass. Thus, in terms of growth, reproduction is an expensive activity.     

The effect of ocean acidification and temperature on the fertilization and embryonic development of the Sydney rock oyster Saccostrea glomerata (Gould 1850)

This study investigated the synergistic effects of ocean acidification (caused by elevations in the partial pressure of carbon dioxide pCO₂) and temperature on the fertilization and embryonic development of the economically and ecologically important Sydney rock oyster, Saccostrea glomerata (Gould 1850). As pCO₂ increased, fertilization significantly decreased. The temperature of 26 °C was the optimum temperature for fertilization, as temperature increased and decreased from this optimum, fertilization decreased. There was also an effect of pCO₂ and temperature on embryonic development. Generally as pCO₂ increased, the percentage and size of D‐veligers decreased and the percentage of D‐veligers that were abnormal increased. The optimum temperature was 26 °C and embryonic development decreased at temperatures that were above and below this temperature. Abnormality of D‐veligers was greatest at 1000 ppm and 18 and 30 °C (≥90%) and least at 375 ppm and 26 °C (≤4%). Finally prolonged exposure of elevated pCO₂ and temperature across early developmental stages led to fewer D‐veligers, more abnormality and smaller sizes in elevated CO₂ environments and may lead to lethal effects at suboptimal temperatures. Embryos that were exposed to the pCO₂ and temperature treatments for fertilization and embryonic development had fewer D‐veligers, greater percentage of abnormality and reduced size than embryos that were exposed to the treatments for embryonic development only. Further at the elevated temperature of 30 °C and 750–1000 ppm, there was no embryonic development. The results of this study suggest that predicted changes in ocean acidification and temperature over the next century may have severe implications for the distribution and abundance of S. glomerata as well as possible implications for the reproduction and development of other marine invertebrates.     

Induction of phenoloxidase and other immunological activities in Sydney rock oysters challenged with microbial pathogen-associate molecular patterns

This study investigates the effects of two pathogen-associated molecular patterns (PAMPs), LPS and zymosan, on the Sydney rock oyster (Saccostrea glomerata) immune system. Phenoloxidase and phagocytic activities, total and differential haemocyte frequencies, as well as peroxide and superoxide concentrations were measured after the injection of lipopolysaccharide and zymosan. All of the immunological parameters were induced by both PAMPs. Phenoloxidase (monophenolase and diphenolase) and phagocytic activities, as well as the frequencies of phenoloxidase-positive haemocytes, hyalinocytes and granulocytes in the haemolymph, increased within 24 h of PAMP injection. Values for all of these parameters peaked within 48 h of challenge and began to decrease to levels that were indistinguishable from those of controls within 96h. The only exception to this pattern was diphenolase activity, which remained elevated for at least 96 h. Control saline injections that lacked PAMPs also induced responses in most of the parameters measured. However, reactions to saline injections were of far lower magnitude compared to those induced by PAMPs. All of the data suggest that the phenoloxidase and phagocytic systems of oysters are inducible components of the Sydney rock oyster immune system, and that induction is primarily due to increased frequencies of specialised haemocytes in the haemolymph.     

Seasonal variability in feeding behaviour, metabolic rates and carbon and nitrogen balances in the Sydney oyster, Saccostrea glomerata (Gould)

To establish if nutrients limit the growth of bivalves requires information not only on the quality of food available, but also the animals' feeding behaviour and endogenous metabolic demands. We hypothesized that growth of the Sydney rock oyster (Saccostrea glomerata) would vary in response to seasonal changes in food quality rather than quantity. We also predicted that the oysters would show feeding preferences for nitrogen over carbon, and this behaviour would result in carbon/nitrogen (C/N) ratios for ingested and absorbed matter that would be lower than the C/N of both the seston and the oysters' estimated metabolic maintenance requirements. The experiments were done in two phases under natural conditions. In phase 1, feeding behaviour was assessed on a single occasion and the results used to pose hypotheses for testing in phase 2, which included measurements made on three occasions encompassing autumn, winter and spring conditions. Growth rates varied with changes in ambient food quality and not with the concentration of total suspended matter. Feeding behaviour responded to food quality and, in most cases, resulted in nitrogen enrichment. For example, when nitrogen was potentially limiting to growth and/or maintenance, due to high food C/N (July) or high nitrogen demand (March), pre-ingestive selection ensured nitrogen enrichment of ingested matter and C/N ratios of ingested matter which were below the maintenance requirement. However, in November, when endogenous demands indicated an increased requirement for carbon, feeding behaviour resulted in carbon enrichment, an increase in carbon conversion efficiency, and ingested C/N ratios greater than the maintenance requirement. The results support the assertion of variable feeding physiology in oysters, responsive to both exogenous (seasonal differences in carbon and nitrogen availability) and endogenous (cycles of reproduction and growth) factors.