Wayne A. O'Connor

Predicting the Response of Molluscs to the Impact of Ocean Acidification

Elevations in atmospheric carbon dioxide (CO2) are anticipated to acidify oceans because of fundamental changes in ocean chemistry created by CO2 absorption from the atmosphere. Over the next century, these elevated concentrations of atmospheric CO2 are expected to result in a reduction of the surface ocean waters from 8.1 to 7.7 units as well as a reduction in carbonate ion (CO32−) concentration. The potential impact that this change in ocean chemistry will have on marine and estuarine organisms and ecosystems is a growing concern for scientists worldwide. While species-specific responses to ocean acidification are widespread across a number of marine taxa, molluscs are one animal phylum with many species which are particularly vulnerable across a number of life-history stages. Molluscs make up the second largest animal phylum on earth with 30,000 species and are a major producer of CaCO3. Molluscs also provide essential ecosystem services including habitat structure and food for benthic organisms (i.e., mussel and oyster beds), purification of water through filtration and are economically valuable. Even sub lethal impacts on molluscs due to climate changed oceans will have serious consequences for global protein sources and marine ecosystems.

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.

The evaluation of fresh algae and stored algal concentrates as a food source for Sydney rock oyster, Saccostrea commercialis (Iredale & Roughley), larvae

Abstract Six algal species were fed singly and in combination with Pavlova lutheri , in both fresh and concentrated form, to 1-day-old Sydney rock oyster, Saccostrea commercialis , larvae for 6 days. Although a number of algal species produced high larval length increases, the best fresh diet consisted of P. lutheri and Isochrysis aff. galbana (T-iso). However, when concentrated to a paste and stored for 7–14 days at 4 °C, a combination of P. lutheri and Chaetoceros calcitrans produced greater length increases than any other fresh or stored, single or combined diet tested. Centrifugation and storage of algae prior to feeding had varied effects on the algal species tested, but it had no detected deleterious effects on any of the diatoms tested.

Salinity and temperature tolerance of embryos and juveniles of the pearl oyster, Pinctada imbricata Röding.

Abstract The responses of embryos and juveniles of the pearl oyster, Pinctada imbricata, to variation in temperature and salinity were investigated to assist hatchery production and farming. Embryos were incubated at temperatures in the range 14–26 °C and salinities in the range 11–35 ppt. Embryos did not develop to D-veliger stage at 14 °C and at salinities of 26 ppt or less. Within the salinity range 29–35 ppt, the percentage embryos developing to D-veliger stage increased significantly with increasing salinity. Within the temperature range 18–26 °C, increasing temperature increased the rate of development as well as the number of embryos developing to D-veliger stage within 40 h. Juvenile P. imbricata (17 mm shell height) held at temperatures in the range of 14–24 °C were exposed to salinities in the range of 11–35 ppt. Spat formed byssal attachments most rapidly at salinities of 29 and 32 ppt, irrespective of temperature. At these salinities, >70% of oysters formed byssal attachments to the aquaria walls within 6 h. Outside this narrow salinity range, the rate of byssal attachment decreased and ceased altogether at salinities of 17 ppt or less. Temperature also affected byssal attachment although the impacts were not as great as those of salinity. Within the optimal salinity range (29–32 ppt), the rate of byssal attachment was fastest at 18 °C, where up to 80% of oysters had attached within 4 h. This rate was slightly greater than that observed at 22 °C, which in turn exceeded those observed at 14 and 26 °C. Salinity and temperature also affected survival. Irrespective of temperature, survival was high at salinities of 32 and 35 ppt. By contrast, high rates of mortality occurred within 7 days at salinities of 23 ppt or less. Onset of mortality was most rapid and overall mortality highest at the two extremes in temperature tested, 14 and 26 °C.

The evaluation of twelve algal species as food for juvenile Sydney rock oysters Saccostrea commercialis (Iredale & Roughley)

Juvenile Sydney rock oysters (Saccostrea commercialis) were fed diets composed of either one of twelve algal species or one of eleven algal species in combination with Skeletonema costatum. When used as the sole diet for 21 days, the diatoms Skeletonema costatum, Chaetoceros calcitrans. Chaetoceros gracilis and Thalassiosira pseudonana produced the greatest live weight increase in spat. When Skeletonema costatum was fed in combination with the remaining eleven algal species for 19 days, diets including Chaetoceros gracilis, Tetraselmis chui and Tetraselmis suecica produced the highest live weight increases. These findings indicated differences in the food value of several algal species to larvae and spat of the Sydney rock oyster.

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.

Induction of anaesthesia in the commercial scallop, Pecten fumatus Reeve

Abstract Anaesthetic properties of a range of chemical compounds were tested on mature commercial scallops ( Pecten fumatus ) as a means of reducing stress and subsequent unplanned spawning caused by routine handling and assessment of breeding condition. Of 14 compounds tested, only chloral hydrate, magnesium chloride and magnesium sulphate successfully induced anaesthesia within 1 h. Magnesium sulphate induced high post-anaesthesia mortality and was not investigated further. Doses of 4 g chloral hydrate/1 (0.024 M) or 30 g magnesium chloride/1 (0.31 M) were selected as most suitable on the basis of time to, and recovery from, anaesthesia. Neither anaesthetic caused mortality nor increased spawning activity and magnesium chloride actually reduced the incidence of unplanned spawning. With the use of chloral hydrate, time to anaesthesia was found to decrease significantly with increasing water temperature in the range 12–24 °C, but to be independent of temperature in the case of magnesium chloride.

Temperature and nutrition as factors in conditioning broodstock of the commercial scallop Pecten fumatus Reeve

Abstract Difficulty in obtaining ripe broodstock of the commercial scallop Pecten fumatus from wild populations in New South Wales prompted the development of hatchery conditioning protocols. Microalgal diets, feeding rates and holding temperatures conducive to rapid gonadal growth and development were identified. Results of microalgal clearance rate experiments indicated that the species Pavlova lutheri, Tahitian Isochrysis aff. galbana, Chroomonas salina and Chaetoceros gracilis are ingested by adult scallops at similar rates at 14, 18 and 21 °C, but more slowly at 11 °C. Maximum (satiation) ingestion rate using mixed diets containing approximately equal numbers of cells of these four species, was estimated as about 6 × 109 cells per day for broodstock in the range 55 to 75 mm shell height. Large numbers of cells of the microalga Tetraselmis suecica passed through the digestive tract of scallops undigested. Egg production rate, gonad size and condition factors were all highest when broodstock were held at 15 °C, lowest for scallops maintained at 21 °C and of intermediate values at temperatures of 12 and 18 °C. Gonad condition and egg production increased as feeding rates were raised from 12.5 to 100% of satiation at all test temperatures in the range 12 to 21 °C. Frequent spawning triggered by routine handling of broodstock during these experiments emphasised the need for conditioning equipment and protocols that minimise handling and other disturbance factors. No unplanned spawning occurred at feeding rates of 25% or 12.5% of satiation at 12 °C. The use of low temperature and low feeding rate might therefore enable short term stockpiling of broodstock once they attain prime breeding condition. Findings of these experiments were successfully applied to a subsequent large scale conditioning and induced spawning trial.

Differential proteomic responses of selectively bred and wild‐type Sydney rock oyster populations exposed to elevated CO2

Previous work suggests that larvae from Sydney rock oysters that have been selectively bred for fast growth and disease resistance are more resilient to the impacts of ocean acidification than nonselected, wild‐type oysters. In this study, we used proteomics to investigate the molecular differences between oyster populations in adult Sydney rock oysters and to identify whether these form the basis for observations seen in larvae. Adult oysters from a selective breeding line (B2) and nonselected wild types (WT) were exposed for 4 weeks to elevated pCO2 (856 μatm) before their proteomes were compared to those of oysters held under ambient conditions (375 μatm pCO2). Exposure to elevated pCO2 resulted in substantial changes in the proteomes of oysters from both the selectively bred and wild‐type populations. When biological functions were assigned, these differential proteins fell into five broad, potentially interrelated categories of subcellular functions, in both oyster populations. These functional categories were energy production, cellular stress responses, the cytoskeleton, protein synthesis and cell signalling. In the wild‐type population, proteins were predominantly upregulated. However, unexpectedly, these cellular systems were downregulated in the selectively bred oyster population, indicating cellular dysfunction. We argue that this reflects a trade‐off, whereby an adaptive capacity for enhanced mitochondrial energy production in the selectively bred population may help to protect larvae from the effects of elevated CO2, whilst being deleterious to adult oysters.

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.