Cryopreservation of gametes is a valuable tool for the fast-growing aquaculture industry in New Zealand. In the present study, research was aimed to improve the cryopreservation of Pacific oyster (Crassostrea gigas) oocytes. For this, two main approaches were used: the modification of an existing published (standard) cryopreservation protocol for oyster oocytes and the modification of the oocytes themselves prior to cryopreservation. The objectives in the chapters of this thesis were: (a) determination of the cryobiological characteristics of oyster oocytes; (b) assessment and reduction of intracellular ice formation (IIF) in oocytes; and (c) modification of the lipid fraction (cholesterol and fatty acids) of oocytes prior to cryopreservation. Knowledge of the membrane permeability parameters in response to concentrations of water and ethylene glycol (EG), the influence of temperature upon these parameters, and the osmotic tolerance limits of oyster oocytes were used to develop computer models that simulated the cellular volume changes that oocytes underwent during EG addition and removal. The models predicted that when one part of EG was added in one step to one part of oocyte suspension and equilibrated for 20 min at 20 �C, similar volume changes in oocytes would be obtained, compared to a more complicated multi-step addition method. This method of addition resulted in similar post-thaw fertilization rates to those obtained by using the multi-step addition method, thus reducing oocyte handling. Cryomicroscopy was used to assess the effect of cooling rates and EG concentration on the temperature at which oocytes underwent IIF. It was found that IIF occurred at higher subzero temperatures when fast cooling rates were used (30 and 5 �C min⁻�) and at EG concentrations ranged between 0 and 15%. At a relatively slower cooling rate of 0.3 �C min⁻� and with 10% EG, which are the conditions employed in the standard cryopreservation protocol, no IIF occurred. The steps of the standard protocol that were more likely to cause oocyte damage were identified by evaluating the fertilization rate of oocytes at each step. Results showed that oocytes were most damaged by cooling them to -35 �C and followed by plunging them in liquid nitrogen. Contrary to what had been observed under the cryomicroscope, transmission electron microscopy (TEM) analysis revealed that all oocytes cryopreserved by the standard protocol contained cytoplasmic ice. In addition, it was also observed that oocytes were at two developmental stages when frozen (prophase and metaphase I). These observations prompted the development of alternative cooling programmes aimed to reduce intracellular ice. The effect of cooling rate, plunge temperature and time held at the plunge temperature were thus evaluated, based on post-thaw fertilization rate of oocytes. Overall, neither the cooling rate nor the holding time had an effect on oocyte fertility. However, the plunge temperature had an effect, where oocytes plunged at -60 �C had lower post-thaw fertilization rates than oocytes plunged at -35 �C. Through the slowing of the cooling rate, lengthening of the holding time and lowering of the plunge temperature, it was possible to reduce the amount of ice in the cytoplasm. However, the reduction of intracellular ice did not improve the post-thaw fertilization rate of the oocytes; on the contrary, post-thaw fertilization decreased notoriously. From these results, it can be suggested that oyster oocytes are more likely to be damaged by exposure to high intra and extracellular solute concentration than IIF during cryopreservation. In an effort to modify the lipid content of oyster oocytes prior to cryopreservation and thus, making them more resistant during cryopreservation, oocytes were incubated in solutions that would add or remove cholesterol or in solutions rich in long chain fatty acids (EPA or DHA). Oocytes incubated in cholesterol-rich solutions showed a positive uptake of fluorescently labelled cholesterol and this effect was dose dependent. Nevertheless, this uptake did not improve the post-thaw fertilization rate nor did it increase the total cholesterol content of the oocytes. When oocytes were incubated in non-conjugated or conjugated EPA or DHA, no increase in the proportion of these fatty acids was identified in the fatty acid profiles of whole oocytes and no improvement of the post-thaw fertilization rate was recorded. Given that there was no uptake of fatty acids from the incubation media by the oocytes, a different approach was taken. This involved the supplementation of lipid-rich diets to the oyster broodstock during gametogenesis (cold-conditioning) and vitellogenesis (warm-conditioning). Despite results showing that lipid content and, indeed, fatty acid profile was altered through the diet, the results also showed that fresh oocytes from broodstock fed during cold-conditioning did not show any improvement in their fertilization rates, nor did they benefit from a lipid-rich diet during warm-conditioning. On the other hand, cryopreserved oocytes did have higher post-thaw fertilisation rates when broodstock were fed during cold-conditioning and, although no effect was found from feeding broodstock with either of the lipid-rich diets during warm-conditioning, trends indicated that a diet consisting of fresh microalgae or the commercial supplement Algamac would yield the highest post-thaw fertilization rates. This thesis has furthered the understanding of some of the factors that determine cryosurvival in oyster oocytes and has demonstrated that both physical and biological issues must be taken into consideration for cryopreservation. Specifically, the results in this thesis helped to modify an empirically developed cryopreservation protocol for Pacific oyster oocytes. In addition, the results also showed strong evidence of the survival of oyster oocytes to intracellular ice and highlighted the importance of supplying the broodstock with lipid-rich food during the periods of gamete formation and maturation in order to obtain oocytes that are more amenable to cryopreservation. These benefits could be of significant practical importance and may be extended for the development or refinement of cryopreservation protocols for other shellfish species of commercial importance to the aquaculture industry of New Zealand.

[Truncated abstract] There is a well-documented global decline of seagrass meadows in response to anthropogenic pressures. Transplantation of rhizome fragments into denuded areas has been used to enhance natural recovery but nutrient limitation and poor root growth may contribute to low success rates. Addition of nutrients to sediments has been proposed as a means of enhancing growth and survival of seagrass transplants by alleviating nutrient limitation but there is limited information of the effects of nutrient additions to seagrass transplants, particularly root development and morphology. In addition to nutrient limitation, sulphide accumulation in sediments with high organic matter has been shown to reduce seagrass growth and experimental iron additions have been shown to enhance seagrass growth by buffering the development of reduced conditions in organic rich sediments. This thesis examines responses (growth, morphology and nutrient status) of Posidonia australis and Posidonia sinuosa transplants to nitrogen (N), phosphorus (P) and chelated iron (Fe EDTA) additions, and includes a detailed investigation of root growth and morphology in response to these additions. Experiments were carried out in underwater plots in Oyster Harbour, a sheltered estuarine inlet with seasonal river flow, located on the southern coast of Western Australia. In the first experiment, nutrients (N, P and N+P) and Fe EDTA were added to sediment underlying transplants at the end of summer (March 2005). ... Nitrogen (both N and N+P) addition also reduced overall biomass allocation to roots. Nitrogen concentrations increased with N addition in P. sinuosa only but both species had more negative d15N values with N and N+P addition indicating that added N had been taken up. In contrast, P addition had little effect on root growth, and P concentrations only increased in P. australis. However, tissue concentrations of Zn, Co, and Mo in both species increased with P addition. Roots showed limited morphological variation (total root length, mean root diameter, root fineness, specific root length, surface area) in response to nutrient addition. Combined N and P addition increased lateral root density (branches m-1) but had no effect on other morphological parameters of the root compartment. Iron addition had no effect on P. sinuosa but P. australis transplants significantly reduced root growth and productivity, particularly in spring/summer growth period, due to lower primary and lateral growth rates. Overall, N or P additions did not enhance growth although these nutrients were taken up. Iron additions also had little effect on transplant growth. Seagrass transplants had significant root growth although they appeared not to respond to nutrient or iron addition. Both species produced extensive root systems capable of taking up sediment nutrients, which suggests that root development was not a limiting factor in establishment and growth of transplants in the temperate estuary, Oyster Harbour.

Heavy metals present in high concentrations in aquatic habitats are bioaccumulated within the tissues of intertidal organisms. The chemical analyses of animal tissues and sediments provide an indication of bioavailability of heavy metals in the environment. Monitoring of the coastal pollution using organisms is widely practiced all over the world.Chemical analysis of the tissues of Pacific oyster, Crassostrea gigas, and river sediments were used in this study to monitor the environmental concentrations, of cadmium, copper, lead and zinc of shallow coastal areas near to the river mouths of Mahurangi, Awaruku, Waiake, Taiorahi and Wairau. All of these river mouths are situated along the northeastern coast of Auckland. Each month, during the period of November 2002 to October 2003, three replicate samples of oysters, and sediments were collected from each of the river mouths for analysis. Three additional replicate samples of oysters were collected separately, in each month in order to calculate the condition index of oysters. Oyster tissues and sediments were analysed with Inductively Coupled Atomic Plasma Emission Spectrometer to detect the concentration levels of cadmium, copper, lead and zinc.The highest level of copper and zinc concentrations in the oyster tissues was observed in the river mouth of Wairau. Wairau river mouth receives water from heavily urbanised and industrialised catchments. A higher concentration of cadmium was observed in the oysters of the river mouth of Mahurangi than in the oysters in the other sites. The reason for this difference could be due to the heavy use of cadmium contaminated fertilizers at the pasture lands situated around the Mahurangi estuary. Therefore, the relationship between the land use of the catchments and the degree of pollution of the estuarine habitats could be established from the data obtained from this study. Higher concentrations of heavy metals were found in the sediments of Waiake, Taiorahi and Wairau compared to the sediments of Mahurangi and Awaruku. Significantly higher level of copper was observed in the sediments of Awaruku. However no clear co-relation was found between heavy metal concentration in oysters and in sediments. Variations of the condition of oysters were closely related to seasonal changes of the life cycle of the oysters. No clear relationship was found between the condition of the oysters and the heavy metal concentration of the river mouth habitats.This study provides evidence that Pacific oysters are good organisms to use as bioindicators of environmental heavy metal levels in shallow coastal waters. The results of this study suggest a clear relationship between the heavy metal concentration in river waters and the land use of the catchment areas of those rivers. The results may be useful in management strategies of the northeastern coastal areas of Auckland.

Mass mortalities of farmed Sydney rock oyster, Saccostrea glomerata, have been observed in Australia since the 1970s and are attributed to the paramyxean protozoan parasite, Marteilia sydneyi (etiological agent of QX disease). This opportunistic parasite infects S. glomerata when the oyster’s immune system has been compromised due to one or more unknown transient environmental stressor. Management and prevention of the disease is seriously compromised as neither the risk factors for the disease, nor the complete life cycle of M. sydneyi are currently understood. The future of the rock oyster industry relies on the development of QX-resistant oysters. Selection of S. glomerata for resistance to QX disease over several generations has revealed that QX-resistance is heritable and likely to be controlled by multiple genes. The main focuses of this thesis was to improve our knowledge of the genes involved in immunity of S. glomerata to disease. The transcriptome response of hemocytes isolated from S. glomerata selected over four generations for resistance to QX disease (QXR4) was compared to non-selected control oysters (W-type) using suppression, subtractive hybridization (SSH) and quantitative real-time PCR (qRT-PCR). Our data supported that differences in gene expression due to selection was largely attributable to constitutive differences in transcriptional rate. The basal-expression of an extracellular superoxide dismutase (EcSOD) and small heat shock protein (sHsP) was 3.18- and 2.05-fold higher in QXR4 oysters, respectively (p < 0.5). The basal-expression of peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine (IK) was 2.75- and 1.5-fold lower in QXR4 oysters, respectively (p < 0.5). Expression of EcSOD, Prx6 and IK was measured in S. glomerata in response to injection with a range of pathogen associated molecular patterns (PAMPs) to replicate microbial invasion as QX disease cannot be replicated in the laboratory due to the complex life cycle of M. sydneyi. The expression of IK was induced 2.2-fold in S. glomerata in response to injection with double stranded RNA when compared to control oysters (p < 0.05). However, changes in the expression of EcSOD and Prx6 could not be induced with any of the tested PAMPs (p > 0.5), suggesting the difference in basal-expression of these two genes between QXR4 and W-type oysters would be maintained during infection with M. sydneyi. It was concluded that QXR4 oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H2O2) faster due to the elevated levels of EcSOD and that the H2O2 would reach higher concentrations due to the reduced levels of Prx6 to detoxify it. Attempts were made to understand why the basal expression of EcSOD and Prx6 is different between QXR4 and W-type oysters. Identification of DNA variants within the promoter regions of these genes would provide greater insight into the gene network(s) involved in disease resistance and possibly lead to identification of QX-resistant markers that can be applied to the Sydney rock oyster breeding program. The differential expression of EcSOD and Prx6 is likely to originate from either modifications in transcriptional rate or mRNA stability as the percentage of hemocytes that express these two genes was shown to be equal between QXR4 and W-type oysters using in-situ hybridization (p > 0.5). Attempts to amplify and assemble the full-length EcSOD gene from S. glomerata were unsuccessful. The full Prx6 gene was amplified and the frequency of polymorphisms that affected mRNA stability and transcriptional rate were determine between QX-resistant and –susceptible S. glomerata (N = 15). Notably, the frequency of a single nucleotide polymorphism (SNP) in the promoter region (-240A>G) affected the binding of a heat shock factor. The genotypic frequency of -240G/G was 0.400 in resistant oysters compared to 0.067 in susceptible oysters (p = 0.059). Further validation of this SNP is now required using a larger data set. Monitoring survival and histological observations of S. glomerata over the 2006/2007 and 2007/2008 QX disease risk period in the Pimpama River, SE Queensland provided further support to anecdotal evidence that mortality of S. glomerata occurs after heavy summer rainfall in SE Queensland. It is presumed that heavy rainfall causes the immune system of S. glomerata to become compromised, presumably by a reduction in salinity and/or estuarine acidification from a rising water table leading to leaching of acid sulfate soils. Laboratory trials revealed a drop in salinity from 35 ppt to 15 ppt affected immune gene expression and inhibition of the Prx6 gene could still be detected five days after oysters were returned to normal seawater. Acid sulfate soil leachate had no effect on the expression of immune genes or immunological parameters tested. This result provides further support to existing evidence that reduced salinity causes the immune system of S. glomerata to be compromised, possibly resulting in higher mortality of S. glomerata when subsequently challenged with M. sydneyi. Examination of S. glomerata during the QX disease risk period revealed oysters were often infected with other parasites. The microsporidian parasite, Steinhausia sp. was frequently observed infecting the gonad tissue of female S. glomerata in histological sections. This infection resulted in hemocytic infiltration and re-absorption of gonad tissue, possibly resulting in reduced growth rates, condition index and marketablity. The frequency of this parasite in oyster samples taken from Moreton Bay, SE Queensland, suggests this parasite could be an emerging problem for oyster farmers in SE Queensland. Differences in the bacterial community within the digestive gland of S. glomerata infected and un-infected with M. sydneyi was observed using non-culture techniques. Healthy oysters had a diverse bacterial community with 23 different operational taxonomic units (OTUs) identified. In contrast, S. glomerata infected with M. sydneyi had only one OTU present in the digestive gland, which was closely related to a Rickettsiales-like prokaryote (RLP) based on phylogenetic analysis of its 16S rDNA sequence. This RLP may be detrimental to its host during concurrent infection with M. sydneyi and warrants further investigation. Overall, this project demonstrated resistance of S. glomerata to M. sydneyi is likely to involve constitutive differences in gene expression. Identification of DNA variants within the promoter regions of differentially expressed genes may provide further insight into gene regulation within oysters and allow identification of DNA markers for selecting QX-resistant brood-stock. Results presented in this thesis support anecdotal evidence that S. glomerata are more susceptible to M. sydneyi following periods of high rainfall as a result of reduced salinity compromising the immune system of S. glomerata. Histological observation of S. glomerata over the QX-disease risk period revealed that oysters were often infected with a range of other parasites. The presence of these parasites may also compromise the immune system of S. glomerata during the QX-disease risk period. The implications of their presence must be factored in to future breeding and research programmes.

Thesis (Ph.D.) - James Cook University, 2004. / Typescript (photocopy) Bibliography: leaves 194-210.

The Olympia oyster, Ostrea lurida. was overharvested in the early 20th century and is now the focus of restoration efforts in estuaries along the west coast of North America. These efforts would be aided by a better understanding of larval abundance patterns, settlement behavior, and post-settlement performance of oysters in estuaries throughout its range. In Coos Bay, Oregon, all three of these components of the oyster life cycle were investigated at multiple sites. Like adult oysters, larvae were restricted to the upper portion of the bay, although larvae were supplied to sites in the upper bay where settlement was low. Settlement and post-settlement growth was highest at sites of high adult density. These results indicate that in O. lurida, as in many other marine invertebrates, the adult population is subject to bottlenecks at the larval and juvenile stage that can vary spatially. This thesis contains previously unpublished co-authored material. / 2014-12-16

Made available in DSpace on 2015-12-10T14:22:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-06-26. Added 1 bitstream(s) on 2015-12-10T14:28:19Z : No. of bitstreams: 1 000854807.pdf: 1505001 bytes, checksum: 3eee682ea18dfba9f3d5ffbd7dff83c1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O cultivo de moluscos bivalves filtradores como as ostras é frequentemente citado como atividade aquícola sustentável. Porém, não há avaliações que quantifiquem a sustentabilidade ambiental desses cultivos. Assim, o objetivo deste trabalho foi avaliar a sustentabilidade ambiental de um sistema de produção de ostras-do-mangue (Crassostrea spp.), com sementes coletadas na natureza, em Cananeia-SP, utilizando conjuntos de indicadores. Foram calculados indicadores para refletir o uso de recursos naturais, a eficiência no uso destes recursos, a liberação de poluentes e a conservação da diversidade genética e biodiversidade. O cultivo usa pouco espaço e pouca água, praticamente não depende da adição de nitrogênio e fósforo e apresenta potencial para retirar esses nutrientes em excesso no ambiente aquático, além de absorver gases causadores do efeito estufa. Além disso, utiliza espécie nativa e local. Deste modo, é possível afirmar que o sistema de produção de ostras de Cananeia apresenta elevada sustentabilidade ambiental / Farming of filter feeding bivalve molluscs such as oysters is frequently cited as a sustainable activity. However, there is no quantitative assessment to support this fact. The aim of this work is to evaluate the environmental sustainability of a mangrove oyster (Crassostrea spp.) production system, with seeds collected in the wild, in Cananeia-SP, Brazil using sets of indicators. The indicators were calculated to reflect the use of natural resources, the efficiency in the use of these resources, the release of pollutants and the conservation of genetic diversity and biodiversity. The culture use low space and water, does not depend on the addition of nitrogen and phosphorous and shows potential to remove surplus nutrients in the aquatic environment and absorb greenhouse gases. In this way, we concluded that the oyster production system of Cananeia showed high environmental sustainability

Orientador: Wagner Cotroni Valenti / Banca: Antonio Fernando Monteiro Camargo / Banca: Fabiana Garcia Scaloppi / Resumo: O cultivo de moluscos bivalves filtradores como as ostras é frequentemente citado como atividade aquícola sustentável. Porém, não há avaliações que quantifiquem a sustentabilidade ambiental desses cultivos. Assim, o objetivo deste trabalho foi avaliar a sustentabilidade ambiental de um sistema de produção de ostras-do-mangue (Crassostrea spp.), com sementes coletadas na natureza, em Cananeia-SP, utilizando conjuntos de indicadores. Foram calculados indicadores para refletir o uso de recursos naturais, a eficiência no uso destes recursos, a liberação de poluentes e a conservação da diversidade genética e biodiversidade. O cultivo usa pouco espaço e pouca água, praticamente não depende da adição de nitrogênio e fósforo e apresenta potencial para retirar esses nutrientes em excesso no ambiente aquático, além de absorver gases causadores do efeito estufa. Além disso, utiliza espécie nativa e local. Deste modo, é possível afirmar que o sistema de produção de ostras de Cananeia apresenta elevada sustentabilidade ambiental / Abstract: Farming of filter feeding bivalve molluscs such as oysters is frequently cited as a sustainable activity. However, there is no quantitative assessment to support this fact. The aim of this work is to evaluate the environmental sustainability of a mangrove oyster (Crassostrea spp.) production system, with seeds collected in the wild, in Cananeia-SP, Brazil using sets of indicators. The indicators were calculated to reflect the use of natural resources, the efficiency in the use of these resources, the release of pollutants and the conservation of genetic diversity and biodiversity. The culture use low space and water, does not depend on the addition of nitrogen and phosphorous and shows potential to remove surplus nutrients in the aquatic environment and absorb greenhouse gases. In this way, we concluded that the oyster production system of Cananeia showed high environmental sustainability / Mestre

La baie de Marennes Oléron, premier site ostréicole français, est influencée par la pollution polymétallique historique de l’estuaire de la Gironde avec des concentrations en cadmium dans les huîtres proches de la limite de consommation européenne (RNO 2006; 5 μg.g-1 ps, ECNo.466/2001). Ces travaux de recherche pluridisciplinaires ont pour objectif de caractériser le comportement des ETM en zone côtière et les mécanismes de contamination en ETM des huîtres, spécifiquement en Cd, dans la baie de Marennes Oléron. Pour cela, sept missions océanographiques en période contrastée ont permis de caractériser la spéciation des ETM à l’embouchure des estuaires de la Charente et de la Gironde ainsi qu’au Pertuis de Maumusson.Une étude spatio-temporelle complémentaire des sédiments de surface de la baie de Marennes Oléron a montré un enrichissement des sédiments de surface en Cd dans la zone sud baie,confirmant la connexion des eaux girondines et l’apport en Cd particulaire à la baie par le Pertuis de Maumusson. Cette zone sud a ainsi été choisie pour mener une transplantation d’huîtres pendant trois mois. L’hydrodynamique régionale, observée par imagerie satellite, a présenté unrôle important dans la distribution et la variation temporelle des concentrations en Cd dissous et particulaires minérales de la baie. La bioaccumulation en Cd des différents organes d’huîtres cultivées sur table a été plus importante que celle des huîtres cultivées directement sur le sol,suggérant le faible rôle de la diffusion de Cd par la remise en suspension des sédiments de surface et du microphytobenthos sur la bioaccumulation. De plus, le temps d’immersion étant relativement proche entre les deux conditions, nous suggérons que la voie trophique via le plankton pélagique participe à la contamination des huîtres en plus de la voie directe. Cette contribution de la voie trophique a été confirmée lors d’expérimentations en conditions contrôlées en laboratoire par le développement d’une méthode de traçage simultané des voies de contamination directe et trophique par ajouts d’isotopes stables de Cd, conduites pour des concentrations 10 fois supérieures à l’environnement et des concentrations réalistes observées en baie de Marennes Oléron (40 ng.l-1 et ~0.7 mg.kg-1). / The Marennes-Oléron Bay, hosting the largest oyster production in France, is influenced by thehistoric polymetallic pollution of the Gironde Estuary, with cadmium levels in oysters close tothe consumption limit level (5 μg.g-1 dw, EC No.466/2001). The aim of this pluridisciplinarywork was to characterize the behaviour of trace metals in the coastal zone and the mechanisms ofCd contamination in oysters in the Marennes Oléron Bay. Seven oceanographic cruises wereconducted during contrasting season to characterize trace metals behaviour and speciation in theGironde and Charente estuaries and the coastal zone. Then, a spatial and temporal study of tracemetals in the surface sediments of the Marennes-Oléron Bay showed punctual Cd-enrichedsediments in the southern part, reflecting the connexion with the Gironde waters and theparticulate Cd inputs via the Maumusson inlet. Thus, this area was chosen to study Cdbioaccumulation in oysters over a three months transplantation. The regional hydrodynamic,observed by satellite images, played an important role on Cd speciation and the temporalvariability of dissolved and particulate Cd concentrations. Cadmium bioaccumulation in organsof oysters reared on tables at 60 cm height was more important than in oysters reared near thesediment, suggesting the absence of Cd released during tidal suspension from sediment andmicrophytobenthos. Furthermore, as the immersion time was closed between the two rearingconditions, we suggested Cd bioaccumulation via the direct pathway and also via trophicpathway of contamination by pelagic plankton ingestion. This trophic pathway of Cdcontamination was validated during laboratory experiments using a simultaneous tracing of Cddirect and trophic pathways in oysters by stable isotope spikes at concencentrations 10-foldhigher than the Gironde Estuary and at realistic concentrations observed in the Marennes-OléronBay.

The primary purpose of this study was to evaluate the feasibility of controlling oyster drills in Tomales Bay, particularly Urosalpinx cinerea and Ocenebra Japonica (astropods, Muricidae), with chemicals which have been succesfully tested on a number of oyster pests, Including urosalpinx, in Long Island Sound (Loosanoff , 1956- 1961). The problem was polo to provide information. en the present status of drills alle is the bay, a basic preliminary in evaluating others of pest control.