Natural Oysters Research Articles

Microplastic accumulation in oysters: Insights from aquaculture and laboratory conditions

Microplastics, emerging pollutants, are present in seafood and pose a potential health risk to consumers. The objective of this study was to assess microplastic concentrations in the surface water, sediments, and oysters of the Cha Va River, an aquaculture production area in southern Vietnam. Laboratory experiments were also conducted to examine rates of ingestion by and bioconcentration of microplastics in oysters. Microparticles including microplastics were measured in all samples collected from the field, and presented 9–27 items m−3 in surface water, 3300–8000 items kg−1 dry weight in sediments, and 0.67–8.33 items ind−1 or 0.02–0.33 items g−1 wet weight in oysters. Fibre shapes and blue colour were the most common while polypropylene, polyester, polyethylene, and polyethylene terephthalate acrylate, alkyd were identified. The presence of microparticles in surface water and sediment was influenced by the complex vertical motion characteristic of tidal environments whereas the microparticles in natural oysters seemed to be closer related to those in sediment than in the surface water. Laboratory experiments showed that oysters incubated in 10 and 1000 microplastics L−1 tanks accumulated 4.1 ± 2.0 and 19.0 ± 11.0 microplastics per individual, respectively. However, bioconcentration of microplastics in oysters was higher (12.37 ± 6.95) in tanks with lower microplastic concentrations than in tanks with higher microplastic concentrations (0.62 ± 0.35). It is a high potential human health risk to the local oyster-consumers because of the annually microparticle ingestion from 657 to 3942 items per person. We suggested further studies on the severity of this potential health risk and policies for minimising microplastic emissions into the environment in Vietnam.

Diversities and Shifts of Microbial Communities Associated with Farmed Oysters (Crassostrea gigas) and Their Surrounding Environments in Laoshan Bay Marine Ranching, China.

Pacific oysters (Crassostrea gigas) are widely cultured in Chinese marine ranching with high economic value. However, mass death of farmed oysters has occurred frequently in recent years because of diseases and environmental disturbance (e.g., high temperatures). In order to analyze the potential relationships between microorganisms and the death of farmed oysters, we compared the dynamics of bacterial and protist communities in oysters at different growth phases using high-throughput sequencing. The results showed that the microbial communities in farmed oysters significantly changed and were markedly different from microbes in natural oysters and the surrounding environments. The number of biomarker taxa among farmed oysters and their surrounding environments decreased gradually with the growth of oysters. During the mass death of farmed oysters, the microbial communities' abundance of ecological function genes changed, and the correlations among microorganisms disappeared. These results enrich our understanding of the dynamics of microbial communities in farmed oysters at different growth phases, illustrating the characteristics of interactions among microorganisms during the mass death of farmed oysters. Our study is beneficial to promote the healthy aquaculture of oysters.

A Longitudinal Study on Enteric Virus Contamination in Bivalves along the Coast of Ibaraki Prefecture, Japan

During the 2014 to 2018 seasons, we conducted a longitudinal study involving enteric virus surveillance in bivalves, including natural oysters and clams harvested in Ibaraki Prefecture, Japan. Some norovirus (NoV) contaminations were detected in natural oysters, whereas no enteric virus was found in clams. NoVs detected in oysters were of the genotypes GII.4 and GII.6, both of which are closely related genetically to the NoV strains prevalent in humans. We found low level of enteric virus contamination in bivalves collected along the coast of Ibaraki Prefecture. The possibility of food poisoning caused by these viruses appears low, and few cases of infectious disease have been observed in the surrounding area. The harvest timing was more related to contamination quantity than the harvest area in many enteric viruses. Our results highlight that contamination of bivalves by enteric viruses may depend upon the prevalence of human diarrhea and illness.

Survey of Gymnophalloides seoi Metacercariae in Natural and Cultured Oysters from Several Western Coastal Areas, Korea.

Gymnophalloides seoi (Digenea: Gymnophallidae) is a human intestinal trematode contracted by eating raw oysters (Crassostrea gigas) in the Republic of Korea (=Korea). It has been known to be highly endemic in Aphae Island, Shinan-gun, Jeollanam-do (Province). However, recent epidemiological status of G. seoi has not been reported since the 1990s. In this study, we investigated the prevalence of G. seoi metacercariae in natural and cultured oysters collected from 3 islands and 2 coastal areas in western parts of Korea. The oysters were examined using the artificial digestion method followed by stereomicroscopy. The overall positive rate of G. seoi metacercariae in natural oysters was 66.0% (99/150), and the oysters collected from Yubu Island showed the highest infection rate (74.0%). However, the metacercarial density per oyster was relatively low (1.5–2.4 per oyster). By contrast, no metacercaria was found in cultured oysters purchased from 2 coastal areas in Chungcheongnam-do. Thus, we could confirm that natural oysters produced from 3 western coastal islands are infected with G. seoi metacercariae, whereas cultured oysters purchased from 2 coastal areas were free from infection.

Ultrafiltration for environment safety in shellfish production: Removal of oyster gametes in hatchery effluents

Triploid oysters are favoured by the aquaculture industry because of their sterility, which results in two advantages: a faster growth than natural oysters and a constant quality throughout all the year, which is attractive for consumers. In France, these oysters are mainly produced by mating natural and tetraploid oysters whose production poses a risk for marine environmental biodiversity if biological material is released into the environment. Therefore, effluents from farms which could produce those kinds of oysters must be treated. The objective of this work was to treat shellfish hatchery effluents by ultrafiltration. The retention of gametes by the membrane, was validated for different scenario, 5 log removals were obtained, and their viability was evaluated after treatment highlighting a destruction of these species after air-backwash. The sustainability of the process facing this organic pollution on the duration of each test but also on the period of the study was demonstrated. A protection of the marine environment biodiversity is obtained with ultrafiltration processes.

Qualidade microbiológica de ostras (Crassostrea sp) e de águas coletadas em cultivos e em bancos naturais de Cananéia (SP)

The consumption of marine organisms, especially bivalve molluscs, might affect the human health, once that they concentrate in their tissues, suspended particles, including pathogenic microorganisms. Cananeia region is the largest oyster producer in the state of Sao Paulo, and in 1999 was founded the Cooperostra (Cooperative), which promotes the oysters depuration process in sterile tanks, leading to reduce and/or eliminate retained substances in their tissues. This study aims (i) evaluate the water quality in the adjacencies of the natural oysters’ beds and during the steps of the depuration process, through the determination of Escherichia coli, total coliforms and thermotolerant coliforms, and (ii)evaluate the contamination of Crassostrea sp by Salmonella sp., total coliforms, thermotolerant coliforms and Staphylococcus coagulase positive, in samples collected at Cooperostra and in natural oysters’ beds, assessing the contamination level of the organisms and the efficiency of the depuration process. The results show a reduce of Salmonella sp and coliforms densities in the oysters’ tissues after the depuration process. However, Staphylococcus coagulase positive shown high levels , which may be indicative of handling contamination. The Salmonella sp results were in agreement with RDC n12.

Qualidade microbiológica de ostras (Crassostrea sp) e de águas coletadas em cultivos e em bancos naturais de Cananéia (SP)

O consumo de organismos marinhos, especialmente moluscos bivalves, pode afetar a saúde humana, uma vez que os mesmos concentram em seus tecidos, partí­­culas em suspensão, incluindo microrganismos patogênicos. A região de Cananéia é a maior produtora de ostras do Estado de São Paulo, e em 1999 foi fundada a Cooperostra (Cooperativa dos Produtores de ostras de Cananéia), que promove o processo de depuração das ostras em tanques estéreis, levando a redução e/ou eliminação das substí­¢ncias retidas nos seus tecidos. Os objetivos do presente estudo foram (i) avaliar a qualidade das águas nas adjacências de bancos naturais e nas etapas do processo de depuração realizado na Cooperostra, através da determinação de Escherichia coli, coliformes totais e coliformes termotolerantes e (ii) avaliar a contaminação de Crassostrea sp por Salmonella sp., coliformes totais, coliformes termotolerantes e Staphylococcuscoagulase positiva, em exemplares coletados em áreas naturais de cultivo e na Cooperostra, avaliando o grau de contaminação dos organismos e a eficiência do processo de depuração. Os resultados mostraram uma diminuição nas densidades de Salmonellasp e de coliformes nos tecidos das ostras, após o processo de depuração, entretanto, as densidades de Staphylococcuscoagulase positiva apresentaram ní­­veis elevados, o que pode ser indicativo de contaminação por manipulação. Os resultados para Salmonella sp. estavam em conformidade com a RDC nº 12.

Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB

A SYBR Green™ I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23.6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 105 colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84.27 ± 1.7 °C; spvB, 88.76 ± 1.0 °C; and 16S rRNA gene, 87.16 ± 0.8 °C).The sensitivity of detection was 10 pg purified DNA (invA) or 105 CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 106 and 102 CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health.

Multiplexed real-time PCR amplification of tlh, tdh and trh genes in Vibrio parahaemolyticus and its rapid detection in shellfish and Gulf of Mexico water

In this study, we have developed a SYBR Green I-based real-time multiplexed PCR assay for the detection of Vibrio parahaemolyticus in Gulf of Mexico water (gulf water), artificially seeded and natural oysters targeting three hemolysin genes, tlh, tdh and trh in a single reaction. Post-amplification melt-temperature analysis confirmed the amplification of all three targeted genes with high specificity. The detection sensitivity was 10 cfu (initial inoculum) in 1 ml of gulf water or oyster tissue homogenate, following 5 h enrichment. The results showed 58% of the oysters to be positive for tlh, indicating the presence of V. parahaemolyticus; of which 21% were positive for tdh; and 0.7% for trh, signifying the presence of pathogenic strains. The C(t) values showed that oyster tissue matrix had some level of inhibition, whereas the gulf water had negligible effect on PCR amplification. The assay was rapid (approximately 8 h), specific and sensitive, meeting the ISSC guidelines. Rapid detection using real-time multiplexed PCR will help reduce V. parahaemolyticus-related disease outbreaks, thereby increasing consumer confidence and economic success of the seafood industry.

Detection of pandemicVibrio parahaemolyticusO3:K6 serovar in Gulf of Mexico water and shellfish using real-time PCR with Taqman®fluorescent probes

We describe a real-time multiplexed PCR method using Taqman probes for the detection of total and pandemic Vibrio parahaemolyticus O3:K6 serovar in oysters and Gulf of Mexico water (gulf water). The specificity of these primers and probes was tested for amplification of a 450 bp thermolabile hemolysin (tlh) and a 369 bp ORF8 amplicon representing all V. parahaemolyticus and post-1996 clinical isolates of pandemic serovar O3:K6, respectively. The sensitivity of detection was 10 pg purified DNA or 10(3) CFU in 1 mL pure culture. Enrichment of this pathogen in oyster tissue homogenate or gulf water for 5 or 8 h resulted in the detection of an initial inoculum of 1 CFU in 1 mL or 1 g of samples. Application of the Taqman PCR assay on natural oysters exhibited a positive detection of V. parahaemolyticus, ranging from 16% to 100% of the samples collected primarily during the summer months. None of the samples exhibited a positive detection of O3:K6 serovar. Rapid and sensitive detection of this pathogen will help shellfish industry and Interstate Shellfish Sanitation Conference (ISSC) undertake appropriate measures to monitor this pathogen in oysters and oyster-growing waters, thereby preventing disease outbreaks and consequently protecting consumer health.

Detection ofVibrio parahaemolyticusin Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 10(4) CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3x10(9) CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (C(T)) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10(3) V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 x 10(3) CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.

A nationwide survey of naturally produced oysters for infection with Gymnophalloides seoi metacercariae.

A nationwide survey was performed to know the geographical distribution of Gymnophalloides seoi (Digenea: Gymnophallidae) metacercariae in Korea, by examining the infection status of locally produced oysters, Crassostrea gigas. A total of 24 coastal areas (myons) of 14 guns ( = counties) in Kyonggi-do, Chollabuk-do, Chollanam-do, Kyongsangnam-do, Kyongsangbuk-do, or Kangwon-do, where natural oysters are produced but G. seoi has never been reported, and 13 areas (myons) of Shinan-gun, Chollanam-do, nearby the known endemic area, were surveyed. Oysters from nonendemic areas were free from G. seoi infection, except Byonsan-myon of Buan-gun, Chollabuk-do, where one of 50 oysters examined was infected with 15 metacercariae of G. seoi. In Shinan-gun, oysters from 10 areas including Aphae-myon ( = township) and Anjwa-myon were infected with the metacercariae, with the infection rate ranging from 1.7% to 100% by areas. The intensity of infection was the highest in Aphae-myon. 785.9 metacercariae per oyster. The results indicate that high prevalence of G. seoi is confined to Shinan-gun, but low grade prevalence is also present in adjacent areas such as Buan-gun, Chollabuk-do.

Effects of GRAS Compounds on Natural Vibrio vulnificus Populations in Oysters

Vibrio vulnificus is a bacterium indigenous to estuarine waters and is known to be a significant human pathogen. Infections are generally associated with the consumption of raw shellfish, especially oysters. We have previously determined that a variety of FDA-approved food preservatives were effective agents in causing lethality of V. vulnificusin vitro. In the present study we tested the effects of these compounds in treating natural oysters and found that diacetyl at concentrations of 0.05% or greater could significantly decrease the number of V. vulnificus naturally present in oysters. Lactic acid and BHA were found not to be effective at concentrations up to 0.05%. We conclude that diacetyl is a possible antimicrobial agent for this organism that might be used in shellfish stock.

Application of PIXE in the study of shellfish

The Bartol in-air Pixe system has been used to study the elemental composition of many varieties of sea life; the sensitivity for the elements from Na to Sr is good, a reasonable spatial resolution is attainable and little preparation of the samples is required. Two examples are cited. First, the composition of the inner nacreous and prismatic regions of mussels ( Mytilus edulis) taken monthly (1981–1982) from the Damariscotta River, Maine, has been measured and, second, the shell and soft tissues of oysters ( Crassostrea virginica) grown under laboratory conditions on substrate of compacted residues from coal-fired power plants have been analysed. The mussel study shows a rather large anomaly in the Sr/Ca ratio for the nacreous region for May which suggests a possible connection with the reproduction cycle. Oysters grown on metal-containing coal-ash substrata gave tissue and shell metal concentrations similar to natural oysters, an indication of no anomalous uptake of metals from the coal-ash substrata.

カキ種苗生産場における生態学的研究-II

Seed oyster (Crassostrea gigas) production at Sendai Bay in well known because its source of supply not only to the domestic but also to foreign markets. Collectig seed oyster in Sendai Bay depends on oyster larvae population in Matsushima Bay, especially those from mother oysters which have an early and large production at spawning. This report deals on the relationship between the appearance of oyster larvae and mother oyster population, and also the relationship between spawning perods and the spawning environment at Matsushima Bay from 1953 to 1970. The following are the results of the survey. 1. A close relationship between the quantity of larvae and that of mother oysters appeared to exist in Matsushima Bay. Therefore mother oysters should be protected in order to stabilize and enlarge the seed oyster population. 2. It was observed that oyster spawnings from spring tide to neap tide was twice as large as those from neap tide to spring tide. 3. It was found that the first highest peak of spawning commenced in the middle of July, the second in late July to early August and the third in early August to middle August. Meanwhile spawnings occurred two to three times every two weeks after the first spawning. 4. The first spawning was found to occur when the water temperature and gravity reached 23.3°C and σ15=20.48, the second spawning 25.1°C and σ15=21.20 and the third spawning 25.7°C and σ15=22.59. However, the quantity of spawning increased from the first to the third. 5. The largest appearance of larvae was found when water temperature and gravity reached 27-28°C and σ15=20.00-23.00. 6. It was obvious that the first spawning was originated from the natural oysters, the second from two years old cultured oysters and the third from one year old cultured oysters. Generally, larvae from later spawnings are more apt to damage because the water temperature falls. Therfore the best way to collect larvae is to procure the first or the second spawning. 7. The author described the characteristics of oyster larvae, supplied from Matsushima Bay to Sendai Bay. Also, techniques of predicting seed collection so as to promote a larger production of the seed has been discussed.