Michael Dove

Identification and characterisation of an ostreid herpesvirus-1 microvariant (OsHV-1 µ-var) in Crassostrea gigas (Pacific oysters) in Australia.

Between November 2010 and January 2011, triploid Crassostrea gigas (Pacific oysters) cultivated in the Georges River, New South Wales, experienced >95% mortality. Mortalities also occurred in wild diploid C. gigas in the Georges River and shortly thereafter in the adjacent Parramatta River estuary upstream from Sydney Harbour. Neighbouring Saccostrea glomerata (Sydney rock oysters) did not experience mortalities in either estuary. Surviving oysters were collected to investigate the cause of mortalities. Histologically all oysters displayed significant pathology, and molecular testing revealed a high prevalence of ostreid herpesvirus-1 (OsHV-1). Quantitative PCR indicated that many C. gigas were carrying a high viral load at the time of sampling, while the load in S. glomerata was significantly lower (p < 0.001). Subsequent in situ hybridisation experiments confirmed the presence of a herpesvirus in C. gigas but not S. glomerata tissues, suggesting that S. glomerata is not susceptible to infection with OsHV-1. Naïve sentinel triploid C. gigas placed in the Georges River estuary in January 2011 quickly became infected and experienced nearly 100% mortality within 2 wk of exposure, indicating the persistence of the virus in the environment. Phylogenetic analysis of sequences derived from the C2/C6 region of the virus revealed that the Australian strain of OsHV-1 belongs to the microvariant (µ-var) cluster, which has been associated with severe mortalities in C. gigas in other countries since 2008. Environmental data revealed that the Woolooware Bay outbreaks occurred during a time of considerable environmental disturbance, with increased water temperatures, heavy rainfall, a toxic phytoplankton bloom and the presence of a pathogenic Vibrio sp. all potentially contributing to oyster stress. This is the first confirmed report of OsHV-1 µ-var related C. gigas mortalities in Australia.

IMPACTS OF ESTUARINE ACIDIFICATION ON SURVIVAL AND GROWTH OF SYDNEY ROCK OYSTERS SACCOSTREA GLOMERATA (GOULD 1850)

Abstract Estuarine acidification caused by outflows from acid sulfate soils (ASS), impacts many estuaries in eastern Australia. Affected waters are characterized by low pH and elevated concentrations of metals, particularly iron, aluminum, and manganese. The effects of low pH and elevated metals, associated with ASS-affected water, on adult Sydney rock oysters Saccostrea glomerata have not been previously studied in detail. Most production of Sydney rock oysters occurs in estuaries along the Australian east coast and has significantly decreased over the last 30 y. To investigate poor oyster production in particular areas of the Manning River, New South Wales, Australia, associations between ASS drainage water, estuarine acidification, and Sydney rock oyster survival and growth were studied. This involved a seven-month field study where water quality and oyster survival and growth was measured at seven sites differentially impacted by ASS-affected waters. Estuarine acidification on the Manning River followed periods of heavy rainfall and caused widely fluctuating pH levels, combined with low electrical conductivities and high concentrations of iron and aluminum at experimental sites selected to expose Sydney rock oysters to ASS-affected waters. Hydrologic characteristics and location of experimental sites controlled the extent of impact caused by ASS-affected waters. Sites exposed to ASS-affected waters had a significantly higher (P < 0.001) level of oyster mortality. Mortalities in small oysters were significantly higher (P < 0.05) than mortalities in large oysters. This was attributed to acid-induced shell degradation, which eventually caused perforation of the thinner shells of small oysters. Mortalities of small oysters increased after 42 days of exposure. ASS-affected waters also significantly reduced large and small oyster growth (P < 0.001). Growth of oysters improved and mortalities stabilized at sites impacted by ASS-affected waters in dryer periods because of the improvement in water quality.

The Changing Face of Oyster Culture in New South Wales, Australia

ABSTRACT Oyster farming is one of the oldest aquaculture industries in Australia and, in New South Wales (NSW), its history dates back some 130 y. Like other industries, it has evolved over time, but during the past 5 y, a number of significant changes have occurred. Although Sydney rock oysters (Saccostrea glomerata [SRO]) remain the most important commercial species, the culture of Pacific oysters (Crassostrea gigas), particularly triploids, has increased significantly. Interest in cultivating other commercially important species, such as flat oysters, Ostrea angasi, has also increased. Overall, hatchery-produced oyster seed has become more readily accessible, particularly for S. glomerata, which, prior to 2003, had been largely unavailable to the majority of the rock oyster industry. For both S. glomerata and C. gigas, breeding programs have become an integral part of industry development and have been the primary reason for hatchery seed uptake in NSW. Across the oyster industry, the emphasis placed on the importance of demonstrating environmental sustainability has increased, and both industry and government have been proactive in protecting the estuarine environments in which oyster farming occurs. Collectively, hatchery development, oyster breeding, and environmental research has “spawned” a number of new research initiatives that have increased fundamental oyster research during the past 5 y.

SALINITY AND TEMPERATURE TOLERANCE OF SYDNEY ROCK OYSTERS SACCOSTREA GLOMERATA DURING EARLY ONTOGENY

Abstract Recurrent larval and spat mortality (>80%) occurring in most Sydney rock oyster, Saccostrea glomerata, hatchery-runs since 1980 has prevented reliable commercial hatchery supply of spat and ultimately precluded the industry from accessing stock from breeding programs for faster growth and disease resistance. To overcome larval and spat mortality, the interactive effects of temperature and salinity on early life stages (embryos, larvae, and spat) of S. glomerata were investigated to optimize rearing conditions and thereby improve survival and growth. The early ontogenetic stages of S. glomerata were held at temperatures in the range from 16°C to 30°C and salinities in the range 10–35 ppt. Development of embryos to D-veliger larvae was significantly affected by temperature (P < 0.001), salinity (P < 0.001) and the interaction of these factors. Most rapid embryonic development occurred at a salinity and temperature of 35 ppt and 26°C. Growth of D-veliger, umbonate and pediveliger larvae was also significantly affected by salinity (P < 0.001) and temperature, as was the growth of spat. Salinity had a significant effect (P < 0.001) on D-veliger larvae and spat survival, whereas temperature had a significant effect (P < 0.001) on D-veliger and pediveliger survival. Survival and growth of umbonate larvae were not affected by either salinity or temperature within the range tested. Surface-response plots were also used to examine interactions between salinity and temperature. The optimal temperature for growth of D-veliger larvae was 28°C and for umbonate and pediveliger larvae was 30°C. Greatest length increases for D-veliger and umbonate larvae occurred at the maximum salinity level (34 ppt) whereas the salinity at which this occurred for pediveliger larvae was 26 ppt. Survival of larvae at these optima exceeded 95%. Best spat growth was at a salinity of 35 ppt and a temperature of 30°C. Spat survival at this salinity and temperature combination was 82%. Maximum spat survival was 93% and was measured at a temperature and salinity combination of 23°C and 30 ppt.

HISTOLOGICAL AND FEEDING RESPONSE OF SYDNEY ROCK OYSTERS, SACCOSTREA GLOMERATA, TO ACID SULFATE SOIL OUTFLOWS

Abstract Estuarine acidification, caused by disturbance of acid sulfate soils (ASS) is a problem that affects many estuaries in eastern Australia. ASS outflows have low pH and elevated concentrations of metals, principally iron and aluminum. Most production of Sydney rock oysters Saccostrea glomerata occurs in estuaries along the Australian east coast and estuarine acidification has been implicated in localized declines in oyster production. Estuarine areas recurrently impacted by estuarine acidification have higher levels of oyster mortality and reduced oyster growth compared with sites that are not acidified. Two laboratory experiments were conducted to investigate reasons for poor oyster production at sites exposed to ASS-affected waters. Behavioral response, soft tissue lesions and filtration rates of S. glomerata when exposed to ASS-affected waters were examined. It was found that ASS-affected water altered oysters’ valve movements and significantly reduced filtration rates at pH 5.5. Acidic treatments (pH 5.1) containing 7.64 mg L−1 of aluminum or ASS-affected water caused changes in the mantle and gill soft tissues after short-term (6 h) exposure. Degenerative effects were also caused by iron contained in ASS-affected water. Iron precipitates accumulated on the gills and mantle and were observed in the stomach, intestine, digestive tubules and rectum. Results from this study highlight the rapid deleterious effects of reduced pHs to oysters and the impacts of iron and aluminum contained in ASS-affected waters.

ECOTOXICOLOGICAL EVALUATIONS OF COMMON HATCHERY SUBSTANCES AND PROCEDURES USED IN THE PRODUCTION OF SYDNEY ROCK OYSTERS SACCOSTREA GLOMERATA (GOULD 1850)

Abstract Progress in the Sydney rock oyster Saccostrea glomerata industry, through the adoption of oyster spat selected for faster growth and disease resistance, has been hampered by long-term variability in commercial hatchery spat supply. As part of a broader study to evaluate spat production impediments, the chronic toxicity of substances commonly used in bivalve hatcheries and the effects of handling procedures during early ontogeny (embryo to D-veliger) were evaluated. Among the substances tested, chlorine, Virkon S and Virkon S for Aquaculture (virucidal disinfectants, Antec International Limited, Suffolk, UK), bore water and stored rainwater were found to significantly affect larval development at practically/commercially-relevant concentrations. Toxicity was determined by quantifying embryo-larval development after 48 h exposure and three tests were performed for each substance or procedure. Concentrations of 0.83–1.66 mg L−1 of chlorine in seawater and 0.05–0.5 mg L−1 of Virkon S in seawater significantly decreased the normal development of embryos after 48 h exposure. An EC50 value of 0.76–1.18 mg L−1 for chlorine in seawater and 0.47–1.01 mg L−1 for Virkon S in seawater was derived. The EC50 value for Virkon S for Aquaculture was 0.99–1.12 mg L−1 and this substance caused significant development problems for larvae at a concentration of 0.5 mg L−1 in seawater. Tests that added stored rainwater to seawater had a significant decrease in the percentage of embryos developing to the D-veliger stage at concentrations greater than 1%, whilst no effect was detected at 0.1%. The EC50 value for rainwater was 0.67% to 2.29%. Similarly, bore water added to seawater caused a significant decrease in the percentage of embryos to develop to the D-veliger stage at a concentration of 10% and no effect was observed at 1%. The EC50 value for bore water ranged between 2.3 and 3.7%. Handling procedures for screening fertilized eggs did not significantly decrease development percentage after 48 h incubation time. Likewise, tests conducted with de-ionized water at concentrations up to 10% added to seawater did not significantly reduce the percentage of embryos to develop to the D-veliger stage after 48 h exposure. This study highlights the sensitivity of S. glomerata larvae to surfactants and disinfectants and identified contaminated water sources.

Adult exposure to ocean acidification is maladaptive for larvae of the Sydney rock oyster Saccostrea glomerata in the presence of multiple stressors

Parental effects passed from adults to their offspring have been identified as a source of rapid acclimation that may allow marine populations to persist as our surface oceans continue to decrease in pH. Little is known, however, whether parental effects are beneficial for offspring in the presence of multiple stressors. We exposed adults of the oyster Saccostrea glomerata to elevated CO2 and examined the impacts of elevated CO2 (control = 392; 856 µatm) combined with elevated temperature (control = 24; 28°C), reduced salinity (control = 35; 25) and reduced food concentration (control = full; half diet) on their larvae. Adult exposure to elevated CO2 had a positive impact on larvae reared at elevated CO2 as a sole stressor, which were 8% larger and developed faster at elevated CO2 compared with larvae from adults exposed to ambient CO2. These larvae, however, had significantly reduced survival in all multistressor treatments. This was particularly evident for larvae reared at elevated CO2 combined with elevated temperature or reduced food concentration, with no larvae surviving in some treatment combinations. Larvae from CO2-exposed adults had a higher standard metabolic rate. Our results provide evidence that parental exposure to ocean acidification may be maladaptive when larvae experience multiple stressors.

Longitudinal study of winter mortality disease in Sydney rock oysters Saccostrea glomerata

Winter mortality (WM) is a poorly studied disease affecting Sydney rock oysters Saccostrea glomerata in estuaries in New South Wales, Australia, where it can cause significant losses. WM is more severe in oysters cultured deeper in the water column and appears linked to higher salinities. Current dogma is that WM is caused by the microcell parasite Bonamia roughleyi, but evidence linking clinical signs and histopathology to molecular data identifying bonamiasis is lacking. We conducted a longitudinal study between February and November 2010 in 2 estuaries where WM has occurred (Georges and Shoalhaven Rivers). Results from molecular testing of experimental oysters for Bonamia spp. were compared to clinical disease signs and histopathology. Available environmental data from the study sites were also collated and compared. Oyster condition declined over the study period, coinciding with decreasing water temperatures, and was inversely correlated with the presence of histological lesions. While mortalities occurred in both estuaries, only oysters from the Georges River study site showed gross clinical signs and histological changes characteristic of WM (lesions were prevalent and intralesional microcell-like structures were sometimes noted). PCR testing for Bonamia spp. revealed the presence of an organism belonging to the B. exitiosa-B. roughleyi clade in some samples; however, the very low prevalence of this organism relative to histological changes and the lack of reactivity of affected oysters in subsequent in situ hybridisation experiments led us to conclude that this Bonamia sp. is not responsible for WM. Another aetiological agent and a confluence of environmental factors are a more likely explanation for the disease.

Stable Isotope Analysis of the Contribution of Microalgal Diets to the Growth and Survival of Pacific Oyster Crassostrea gigas (Thunberg, 1979) Larvae

ABSTRACT Stable carbon and nitrogen isotope analyses were used to evaluate the influence of microalgal diet on growth and survival of hatchery-reared Pacific oysterCrassostrea gigas larvae.D-veliger larvae were fedmonospecific diets of Pavlova lutheri (P), Tahitian Isochrysis aff. galbana (T), and Chaetoceros calcitrans (C), and a standard hatchery trispecific diet of 25P:25T:50C (PTC). The dietary effects on nutrient assimilation, isotopic turnover rates, and discrimination factors were assessed using exponential models. Of the four dietary treatments evaluated over the 15-day feeding trial, larvae fed C. calcitrans and PTC had the best growth and high survival. Larvae in all dietary treatments had relatively high isotopic turnover rates (0.075–0.327/day), although some discrimination factors found in this study were out of the normal range for aquatic invertebrates (Δ13C: 0.4‰–4.1‰ and δ15N: 0.1‰–5.3‰). Overall, the turnover rates and discrimination factors for δ13C and δ15N indicated that C. gigas larvae assimilate C. calcitrans and PTC best under hatchery rearing conditions. The results demonstrated the application potential of stable isotope analysis to evaluate optimal diets and feeding regimes to enhance hatchery performance of bivalve larvae and postlarval stages.

Ocean acidification but not warming alters sex determination in the Sydney rock oyster, Saccostrea glomerata

Whether sex determination of marine organisms can be altered by ocean acidification and warming during this century remains a significant, unanswered question. Here, we show that exposure of the protandric hermaphrodite oyster, Saccostrea glomerata to ocean acidification, but not warming, alters sex determination resulting in changes in sex ratios. After just one reproductive cycle there were 16% more females than males. The rate of gametogenesis, gonad area, fecundity, shell length, extracellular pH and survival decreased in response to ocean acidification. Warming as a sole stressor slightly increased the rate of gametogenesis, gonad area and fecundity, but this increase was masked by the impact of ocean acidification at a level predicted for this century. Alterations to sex determination, sex ratios and reproductive capacity will have flow on effects to reduce larval supply and population size of oysters and potentially other marine organisms.