Extraction of DNA,RNA, and glycogen from oysters

The macromolecules DNA, RNA, and glycogen (CHO)_N were extracted with phenol from eggs, sperm, and the combined gill, mantle, and digestive gland tissues of oysters, using methods effective for the HeLa cells. From the eggs, most of the (CHO)_N and RNA coprecipitated in 20% ethanol whereas the DNA precipitated from 50% ethanol solutions. From the combined tissues all three macromolecules precipitated in 20% ethanol whereas from sperm they precipitated from 50% ethanol solutions. The DNA preparation from sperm was viscous and white but contained mostly (CHO)_N and some RNA. However, the DNA-(CHO)_N-RNA preparation was rendered insoluble in saline by CaPO₄ treatment and was not toxic for mammalian kidney cells in tissue culture. Thus, the methods do not yield pure nucleic acids but appear suitable for attempts to extract infectious DNA from oysters.     

Introduction,establishment and expansion of the Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis> in the Oosterschelde (SW Netherlands)

The Pacific oyster Crassostrea gigas was first introduced as an exotic species by oyster farmers in 1964 in the Oosterschelde estuary (SW Netherlands). The initial phase is not well documented but first natural spatfall was recorded in 1975. Excessive spatfall occurred in 1976 and this is considered the start of the expansion phase of the wild oysters. Oyster beds in intertidal and subtidal areas of the Oosterschelde estuary have been growing since. The development in the intertidal area has been reconstructed by using aerial photography, validated by ground truth in 2000–2002. In the subtidal areas extensive oyster beds have been detected by using side scan sonar; on hard substrates along the dikes coverage with oysters up to 90% locally has been recorded by scuba diving surveys. Expansion has also occurred into adjacent water bodies including the Wadden Sea. By forming resistant reefs the oysters induce structural changes in the ecosystem. It is concluded that bed area is still expanding while decrease of the fraction live animals may indicate adjustment of the stock size to the local conditions.     

Ecosystem response to bivalve density reduction: management implications

Coastal ecosystems are easily overexploited and changed by physical and biological factors. In this paper, we discuss current ideas and arguments for coastal ecosystem management with an emphasis on systems that have large bivalve filter feeder components. For centuries the species or population approach has been utilized in fisheries management. With the growing knowledge base on specific environmental effects and relationships, it has become increasingly evident that a broad or holistic approach to fisheries management in these systems is usually more appropriate. An ongoing ecosystem scale experiment in which oysters are completely removed from tidal creeks is described and used as a case study. The experimental design takes estimates of the systems carrying capacity into account. Using the population or species approach to monitor the oysters, the only observable change after the experimental manipulation was a slight increase in summer somatic growth and elevated recruitment of oysters in creeks with oyster reefs removed. These data are interpreted as an indication that the creeks with oysters present are below or near carrying capacity. However, when nekton, plankton and water chemistry data are also examined a much more complicated picture emerges. During the summer growing season, nekton biomass in all creeks is often greater than oyster biomass. Also, our calculations show that oysters do not produce enough ammonium to satisfy phytoplankton productivity, but nekton, water column remineralization and sediments can account for most of the deficit. Finally, microflagellates, which are a preferred food for the oysters, dominate the phytoplankton during the summer growing season and diatoms dominate the colder months. The timing of the change in phase of phytoplankton dominance seems to mirror the seasonal arrival and departure times of nekton in the creeks. We argue that dense bivalve reefs and beds are indicative of intense positive feedback loops that make their ecosystems susceptible to dramatic changes in structure. Such changes have not been reported for natural systems, but are found in systems influenced by over-fishing, nutrient loading and pollution. Thus, the management of sustainable fisheries in coastal ecosystems requires an understanding of the ecosystem science and the realization that systems dominated by bivalves exhibit complex responses that are not easily explained by linear dynamics.     

Development of SNP‐genotyping arrays in two shellfish species

Use of SNPs has been favoured due to their abundance in plant and animal genomes, accompanied by the falling cost and rising throughput capacity for detection and genotyping. Here, we present in vitro (obtained from targeted sequencing) and in silico discovery of SNPs, and the design of medium‐throughput genotyping arrays for two oyster species, the Pacific oyster, Crassostrea gigas, and European flat oyster, Ostrea edulis. Two sets of 384 SNP markers were designed for two Illumina GoldenGate arrays and genotyped on more than 1000 samples for each species. In each case, oyster samples were obtained from wild and selected populations and from three‐generation families segregating for traits of interest in aquaculture. The rate of successfully genotyped polymorphic SNPs was about 60% for each species. Effects of SNP origin and quality on genotyping success (Illumina functionality Score) were analysed and compared with other model and nonmodel species. Furthermore, a simulation was made based on a subset of the C. gigas SNP array with a minor allele frequency of 0.3 and typical crosses used in shellfish hatcheries. This simulation indicated that at least 150 markers were needed to perform an accurate parental assignment. Such panels might provide valuable tools to improve our understanding of the connectivity between wild (and selected) populations and could contribute to future selective breeding programmes.     

Inactivation of Vibrio parahaemolyticus in pure culture, whole live and half shell oysters (Crassostrea virginica) by X-ray

Aims:  To study the inactivation effect of different doses of X-ray on Vibrio parahaemolyticus in pure culture, inoculated whole live and half shell oysters and to evaluate the efficacy of X-ray doses on reduction of inherent microflora on oysters.
Methods and Results:  X-ray was produced using RS 2400 generator system (Rad Source Technologies Inc.). Pure culture of V. parahaemolyticus , inoculated half and whole shell oysters with V. parahaemolyticus were treated with 0·0, 0·1, 0·5, 0·75, 1·0, 1·5, 2·0, 3·0 and 5·0 kGy X-ray. Surviving bacteria in the pure culture and inoculated oysters, before and after treatment, were enumerated using overlay plating (in TSA then TCBS) and most probable number (MPN) methods. A greater than 6·0 log reduction of V. parahaemolyticus was observed with 0·75, 2·0 and 5·0 kGy X-ray for pure culture, half shell and whole shell oysters, respectively. Treatment with 0·75, 2·0 and 5·0 kGy X-ray reduced the MPN to <3 for pure culture, half and whole shell oysters, respectively. Treatment with 1·0 kGy X-ray significantly ( P <  0·05) reduced the inherent micro-organisms on whole shell oysters from 4·7 ± 0·1 to less than the detectable limit (<1·0 log CFU g⁻¹).
Conclusions:  X-ray (1–5 kGy) significantly ( P <  0·05) reduced V . parahaemolyticus and inherent microflora on oysters to less than detectable limit (<1·0 log CFU g⁻¹).
Significance and Impact of the Study:  Treatment with X-ray could control pathogenic bacteria and extend the shelf life of oysters.     

New Resources for Marine Genomics: Bacterial Artificial Chromosome Libraries for the Eastern and Pacific Oysters (Crassostrea virginica and C. gigas)

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at     

Characterization of 27 microsatellite loci in the European flat oyster Ostrea edulis

The flat oyster Ostrea edulis is native to Europe and populations have been severely depleted by the parasite Bonamia ostreae since the 1980s. Additional genetic markers are required to improve population genetics study and linkage map development for selection for B. ostrea-resistance in this species. Here, we characterized 27 novel microsatellite loci for O. edulis. Number of alleles per locus ranged from 6 to 25 and observed heterozygosity between 0.375 and 1. Null alleles were suggested at a few loci but most loci were in Hardy-Weinberg agreement enabling their reliable use in further population and mapping genetics approaches.     

Eleven novel microsatellite markers for the Asian oyster Crassostrea ariakensis

Clones from four partial genomic libraries of the Asian oyster Crassostrea ariakensis were screened to reveal 64 microsatellite-containing sequences. Polymerase chain reaction primers were designed for 34 of these loci. Reactions were optimized for 11 primer pairs and markers were evaluated in eight family crosses and one wild population from northern China. Zero to three null alleles per locus were identified in family crosses, and two of the 11 loci showed allele frequency deviations from Hardy-Weinberg equilibrium in the wild population. These markers have proven useful for genetic studies of C. ariakensis.     

A preliminary study on the effects of oyster culturing structures on birds in a sheltered Irish estuary

This study investigated the species composition, numbers and behaviour of birds in an intertidal oyster culture area in Cork Harbour . These data were compared to a nearby area free of aquaculture within the same estuary in March 1999. Species which occurred in the aquaculture free area were also observed in the trestle-area. The most abundant species were oystercatcher Haematopus ostralegus, redshank Tringa totanus, dunlin Calidris alpina, curlew Numenius arquata, black-headed gull Larus ridibundus and common gull Larus canus. Oystercatcher, curlew, black-headed gull and common gull occurred in significantly lower numbers in the trestle area, while for redshank and dunlin the differences were not significant. The percentage of birds feeding did not differ between the two areas. Oystercatcher, redshank, dunlin and curlew mostly fed in both areas. In contrast, black-headed gull and common gull generally did not feed, but surveyed the area. Whether the trestles were covered by oyster bags or not did not have any effect on the number of birds except for the dunlin. Dunlin were significantly more frequent beneath the trestles with bags compared with those without bags. In general, the percentage of birds feeding did not differ between areas. Interspecies differences occurred with regard to the position occupied by birds at the trestles. Oystercatcher, redshank and curlew spent more time underneath the trestles. Dunlin, black-headed gulls and common gulls did not differ in numbers underneath or on top of the trestles. These preliminary observations at a single time period give some insight as to the potential interactions between shellfish aquaculture and intertidal birds.     

The relationship between lamellar-type cytosomes,DOPA decarboxylase,and lateral ciliary activity of the oyster Crassostrea virginica (Gmelin): Response to salinity change

Lamellar transformations of cytosomes, DOPA decarboxylase and lateral ciliary activity were measured in neuronal components and/or ctenidia of the oyster, Crassostrea virginica (Gmelin). Prolonged exposure to light, anoxic conditions, EGTA (a calcium chelator), and exogenous perfusion of dopamine (a cilioinhibitory neurotransmitter) increases the frequency of lamellar type cytosomes. This is accompanied by decreases in enzymatic activity and a lateral cilioinhibition. Prolonged periods of darkness, electrical stimulation of the branchial nerve and exogenous perfusion of serotonin (a cilioexcitatory neurotransmitter) reduces the conversion of lamellar type configurations, increases enzymatic activity and produces a lateral cilioexcitation. Salinity changes that produce an increase to higher acclimation values result in a gradual decrease in the number of lamellar configurations with a subsequent increase in enzymatic activity and lateral cilioexcitation. The magnitude of the lateral cilioexcitation-cilioinhibition and decarboxylase activity is directly related to the number of lamellar conversions.