Impact of Feeding Status on the Efficacy of Depuration for Decontaminating Vibrio parahaemolyticus in Pacific Oysters (Crassostrea gigas)

Vibrio parahaemolyticus causes acute human gastroenteritis and is often linked to consumption of raw oysters. Previous investigations indicated that refrigerated seawater depuration at 12.5°C could significantly reduce V. parahaemolyticus contamination in Pacific oysters; however, further optimization is necessary to achieve the regulatory target of >3.52 log most probable number (MPN)/g reduction. The current study investigated influences of algal feeding on efficacy of depuration to reduce V. parahaemolyticus in raw oysters. A V. parahaemolyticus cocktail (10290, 10292, 10293, BE 98–2029, 027-1c1) was mixed in artificial seawater (70 L) to inoculate oysters (n = 35) at 4–5 log MPN/g. Inoculated oysters were subjected to depuration with feed (algae = 0.036 ml/gram of oyster) and without feed at 12.5°C. Oysters (n = 5) were analyzed for V. parahaemolyticus using a three-tube MPN method after 0, 1, 3, 5, and 6 days of depuration. Depuration (6 days) achieved average V. parahaemolyticus reductions of 2.75 log MPN/g and 3.03 log MPN/g in the fed and unfed systems, respectively; however, feeding status did not significantly impact the efficacy of depuration to reduce V. parahaemolyticus in Pacific oysters. Further optimization of depuration is necessary to achieve the regulatory target for V. parahaemolyticus decontamination in raw oysters.     

Selection of lactic acid bacteria as candidate probiotics for Vibrio parahaemolyticus depuration in pacific oysters (Crassostrea gigas)

The application of probiotics in food is now widespread and is widely accepted by consumers. Lactic Acid Bacteria (LAB) were isolated from traditional salted fish and then characterized by its ability to inhibit Vibrio parahaemolyticus growth using minimal inhibitory concentration tests. Five out of these strains were identified as Lactobacillus delbrueckii, Lactobacillus casei and three Lactobacillus gasseri by PCR using 16S and 23S rRNA genes. Antibiofilm activity of Lactobacillus spp. extracts were also tested in 96 polystyrene plates. A potential antibiofilm effect was demonstrated as most LAB. Although most LAB extract were able to eradicate pre‐formed biofilm, results demonstrated that five Lactobacillus spp. exhibited a stronger inhibitory effect against V. parahaemolyticus in infected oysters. Vibrio parahaemolyticus viable cells number declined from 10⁶ UFC to 10⁴ UFC after 3 days of incubation with Lactobacillus spp. Probiotic applications, in biological control of seafood associated pathogens can be an alternative solution, providing consumer with a product of good quality owing to the use of non‐toxic compounds. Based on our results, LAB could be used as a bioprotective culture in oyster's depuration to prevent V. parahaemolyticus growth.     

葡萄牙牡蛎的净化及其低温贮藏研究

为保证生食葡萄牙牡蛎(Crassostrea angulata)的安全,在贝水比1∶10、水交换率3次/h、水温28℃、盐度28的条件下,采用流水式紫外线杀菌方法净化48 h,检测净化前后13个卫生指标的变化;净化后的牡蛎在冷藏(4℃)和冷冻(-18℃)两种条件下贮藏7 d以上;检测贮藏过程中菌落总数、大肠菌群和主要营养成分的变化。结果显示,经过48 h净化,菌落总数和大肠菌群含量均大幅下降,重金属、化学污染物等指标变化不大;两种贮藏条件下,菌落总数和大肠菌群都呈先下降再上升的变化,但始终处于安全水平内;蛋白质、脂肪、糖原含量缓慢下降,乳酸含量快速上升。研究表明,葡萄牙牡蛎在紫外线杀菌净化48 h后,至少在冷藏6 d或冷冻11 d内可以保证食品安全。     

Exploration of the antibacterial proteins in pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis> induced by bacterial inoculation

The aim of this research was to characterize immune-related antibacterial substances from pearl oyster Pinctada fucata induced by bacterial invasion. Bacteria inoculation was performed by injecting 0.1 ml of 1.0 × 10¹² colony-forming units/ml Vibrio parahaemolyticus into adductor muscle. Acidic extracts were prepared with 0.1% trifluoroacetic acid from different tissues after 8 h of injection, and antibacterial activity against V. parahaemolyticus was determined via the microdilution broth method. The acidic extracts from gills of inoculated oysters (AEg) showed stronger antibacterial activity than those from non-inoculated ones. Based on this result, antibacterial proteins were purified from AEg via two-step gel filtration chromatography, followed by high-performance liquid chromatography using a TSkgel G3000 column. Protein components were analyzed by both sodium dodecyl sulfate and native polyacrylamide gel electrophoresis. As a result, two antibacterial proteins, APg-1 (with a molecular mass of approximately 210 kDa) and APg-2 (of approximately 30 kDa), were obtained from AEg. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and partial amino acid sequences revealed that these proteins might be novel antibacterial proteins. These results indicate that antibacterial proteins are potentially upregulated in the gill of pearl oysters or released therefrom for defense against bacterial invasion.     

Tetraploid induction in tropical oysters,Crassostrea belcheri (Sowerby) and Crassostrea iredalei (Faustino)

Tetraploid induction has been conducted on temperate oysters but not on tropical oysters. In this study, different heat shocks (32, 35 and 38°C) and cold shocks (1, 4 and 7°C) were used to induce tetraploidy in two tropical oyster species, Crassostrea belcheri and Crassostrea iredalei, through meiosis I inhibition. Temperature shocks were applied on the newly fertilized eggs at 8–10 min post fertilization and terminated when second polar bodies began to form in the control eggs. The ploidy of the larvae and spat was determined via direct chromosome count. The percentage of larval survival until Day 20 was low (between 0.4% and 42.9%) for both temperature shocks and oyster species. No surviving larva was recorded for induction at 1, 4 and 38°C. Tetraploid spat was only recorded in C. iredalei but the percentage is low through heat shock induction of 32 and 35°C. This study shows that the tetraploid induction success rate was slightly higher in C. iredalei compared to C. belcheri. No surviving tetraploid spat were recorded for both oyster species through the cold shock method. This study shows that heat shock can be used to inhibit meiosis for the production of tetraploids but more experiments need to be conducted to determine the optimum temperature when dealing with tropical oysters.     

3种螺对养殖鲍外壳附着牡蛎的防除作用

为研究养殖皱纹盘鲍(Haliotis discus hannai Ino)外壳附着牡蛎的防除方法,对比了疣荔枝螺(Thais clavigera Kuster)、润泽角口螺(Ceratostoma rorifluum)和甲虫螺(Cantharus cecillei)对鲍外壳表面牡蛎的防除效果。以桑沟湾筏式养殖鲍为研究对象,自7月中旬开始,每隔15 d左右分别投放3种螺到鲍养殖笼中,共投放6次,以始终未投放螺的养殖笼为对照组。结果显示,实验结束时甲虫螺组中鲍壳上平均存活牡蛎数目为(0.04±0.04)个/只,疣荔枝螺组和润泽角口螺组疣荔枝螺组分别为(2.49±0.91)个/只和(2.21±1.05)个/只,对照组为(3.33±0.46)个/只。投放3种螺组中鲍壳上存活牡蛎数均显著显著低于对照组(P<0.05),甲虫螺也显著低于其他2种螺(P<0.05)。投螺时间也会影响螺防除牡蛎污损的效果,投放时间过晚会导致螺捕食牡蛎后有明显的壳残留,牡蛎的壳高达到0.5 cm左右再投放螺即可保证防除效果。另外甲虫螺组鲍壳上死亡牡蛎残留壳长度为(0.73±0.27) cm,显著低于疣荔枝螺组...     

Sex-specific growth and condition of the Pacific oyster (Crassostrea gigas Thunberg)

In order to study the possibility of gaining commercial benefit from culturing an excess of one sex of the Pacific oyster (Crassostrea gigas), comparative data on the growth rate and condition of male and female oysters are reported. Historically, measurement of sex‐specific growth rate in oysters has been overlooked or confounded by protandric sex. The recent conclusion that the sex ratio of Pacific oysters is predominantly under genetic rather than environmental control introduces the possibility of manipulating sex ratio for commercial gain if they exhibit asynchronous sex‐specific growth rates. Pacific oysters were cultured intertidally in Smoky Bay, south Australia. The observations, made over the 7‐month gametogenic cycle from August to February to ensure no sex reversal, were of growth rates of male and female oysters and ambient chlorophyll a concentrations. Mean shell growth of female oysters was significantly faster than that of males (4.5 ± 3.3 compared with 3.8 ± 3.2 μm day^?1 mm^?1 total length). Sex‐specific asymmetries in length and weight were generally significant and increased in magnitude during the 7‐month study period, suggesting potential commercial benefits from increasing the proportion of cultured female oysters. The fastest increase in the sex‐specific disparity in growth and condition came after the October chlorophyll a peak, suggesting that females utilize blooms more efficiently than males. Our results compare favourably with methods currently used to increase oyster growth (e.g. triploidy can provide growth gains of 13–51%).     

Growth of the oyster Crassostrea corteziensis (Hertlein, 1951) in Sonora, Mexico

This is the first evaluation of growth and survival of spat of the Cortez oyster Crassostrea corteziensis (Hertlein) produced under controlled conditions in a coastal area in the state of Sonora, Mexico for aquaculture purposes. A suspended culture technique, used for the Pacific oyster C. gigas, was used. The Cortez oyster has an isometric shell growth during the first 13 months, reaching 71.3±1.9 mm length, 52.6±1.3 mm thickness and 25.1±0.8 mm width. Allometric growth was found between total weight and length, thickness and width (survival was 70%). The relationships between particulate organic, inorganic material, chlorophyll a and environmental parameters with growth are described. Growth rates of C. corteziensis were affected by temperature with retardation at less than 18°C. For aquaculture purposes, it is recommended that spat be sowed after winter, and oyster harvest occur at the end of autumn. According to the von Bertalanffy equation, Cortez oysters would reach the traditional exploitation size of 65 mm (mean length) at harvest. Finally, the results of this study have shown that C. corteziensis is a good candidate for aquaculture projects in this region.     

Fouling and predation; how do they affect growth and survival of the blacklip pearl oyster, Pinctada margaritifera, during nursery culture?

This paper reports on 5 experiments conducted to assess the effect of cleaning regime and predation on growth and survival of blacklip pearl oyster (Pinctada margaritifera) juveniles in north Queensland, Australia. P. margaritifera juveniles with a mean (±SE) dorso-ventral shell height (DVH) of 4.5 ± 0.1 mm were placed into plastic mesh trays and cleaned either every 4 or 8 weeks or left uncleaned for 16 weeks. Cleaning regime had a significant effect on growth and survival (P < 0.005). Lowest DVH (16.2 ± 1.0) was shown by oysters in uncleaned trays during 16 weeks compared to oysters in cleaned trays; however, there was no significant difference in DVH between oysters held in trays cleaned every 4 (19.4 ± 1.2) or 8 weeks (21.2 ± 0.8). In contrast lowest survival was shown by oysters held in trays that were cleaned every 4 weeks (30 ± 5%), but no differences were noted between oysters cleaned every 8 weeks (63 ± 4%) and oysters that were left uncleaned for 16 weeks (75 ± 8%). Predators of P. margaritifera in northern Australia included crabs, stomatopods, flatworms, gastropods and fish. The stomatopod, Gonodactylus falcatus, was the most destructive predator with individuals consuming in excess of 20 juvenile pearl oysters per week. The leather jacket, Paramonocanthus japonicus, did not kill pearl oysters, but trimmed the margin of oysters shells significantly reducing DVH when compared to control groups cultured without fish. Removing predators monthly had a significant effect on growth of pearl oysters compared to oysters in non-inspected trays; however monthly inspection of culture trays did not significantly improve oyster survival. This revised version was published online in August 2006 with corrections to the Cover Date.     

Hot water immersion lowers survival,shell growth rate and lysosomal membrane stability of oysters Crassostrea virginica (Gmelin)

This study compares the effect of two anti‐fouling treatments, hot water immersion (15 s at 60°C) and air drying (72 h) on the physiological status of the Eastern oyster Crassostrea virginica. The negative impact of hot water immersion was greater than that of air drying, but varied depending on the initial size of the oysters (40 vs. 60 mm shell height) and the time of the year (June vs. August). Groups treated with hot water exhibited a higher proportion of haemocytes with destabilized lysosomal membranes (HDLM; 47.5 ± 3.1%) than those exposed to air drying (37.5 ± 2.9%). This suggests that the oyster immunocompetency may be lowered by hot water immersion. Overall, the large oysters had lower HDLM values (32.9 ± 3.5%) than the small individuals in June (45.7 ± 2.8%) but similar values in August (46.6 ± 3.5%). Small oysters subjected to hot water immersion in June exhibited a 50% reduction in shell growth and a 50% mortality rate after one month. Our results indicate that air drying is more suitable than hot water immersion as an anti‐fouling treatment for <45 mm oysters.     

The administration of Lactobacillus plantarum directly to food: An effective way to improve the survival and growth of juvenile lined seahorses (Hippocampus erectus)

Previously, we found Lactobacillus plantarum had probiotic effects on lined seahorse (Hippocampus erectus) juveniles by the addition Lactobacillus directly into rearing seawater containing juveniles and their food (i.e. copepods). However, how the juveniles consumed Lactobacillus, directly from water or from copepods in whose guts Lactobacillus may have accumulated by ingestion, is still unclear. To clarify this, three experiments were successively carried out in this study. In the first experiment, the manner by which juveniles consumed Lactobacillus, namely, directly from food, was discovered via a 2 × 2 factorial design with two factors of water and food, and two levels with and without L. plantarum. In the second experiment, a 5 × 4 factorial design with five levels of L. plantarum doses (D: 5, 10, 20, 40 and 80 × 10⁸ cfu/L) and four levels of enrichment time (T: 0.5, 1.0, 1.5 and 2.0 hr) was conducted to analyse lactic acid bacteria (LAB) contents in the enriched copepods under different treatments. In the third experiment, six kinds of Lactobacillus‐enriched copepods chosen from the second experiment that carried significantly different LAB contents were fed to juveniles, and it was found that the copepods containing LAB greater than 5 log₁₀colonies/g had the most improved effects for survival and growth. In conclusion, the effective manner of L. plantarum administration in seahorses is by the addition to their food. Additionally, D40 * T1.0, whose LAB content was more than 5 log₁₀colonies/g, is generally the most economic copepod treatment for improving survival and growth in H. erectus rearing.     

A partial substitution of microalgae with single cell detritus produced from seaweed (Porphyra haitanensis) for the nursery culture of tropical oyster (Crassostrea belcheri)

Oyster seed production in hatchery and nursery systems has relied on the production of microalgae, which is cost effective, but often unpredictable. The development of a satisfactory substitute diet would greatly reduce production costs of hatchery operations. Single cell detritus (SCD) production from seaweed (Porphyra haitanensis) may have great potential as a partial substitute for microalgae. In this experiment, a technique for producing SCD was developed and the product was used as a partial substitute for microalgae for the nursery culture of juvenile tropical oysters (Crassostrea belcheri) in a hatchery setting. A reverse osmosis technique was used on ground seaweed (particle size <200 μm) immersed in freshwater and placed on a shaker at 100 rpm for 2 h before being returned to seawater. This technique was shown to be highly effective for SCD production with a density of 33.7 ± 7.0 × 10⁴ cell mL^?1 and the percentage of SCD particle diameter <20 μm was 89.2%. A partial replacement of microalgae with SCD was found to be unsuitable for nursing oyster larvae. For juvenile oysters (shell width 1.85 ± 0.03 mm and shell length 1.78 ± 0.06 mm) substituting 75% of microalgae with SCD showed lower absolute shell growth, and lower daily yields and survival rates when compared to rates substituting 50% or lower substitution with SCD, or 100% microalgae (P < 0.05). It was found that substituting 50% of the traditional microalgae with SCD produced from seaweed (P. haitanensis) can be used as a partial microalgae substitute for the nursery culture of the juvenile tropical oyster.     

Effect of physicochemical factors on the survival of Vibrio parahaemolyticus on fish

Various physicochemical agents were tested for their effects on the survival of Vibrio parahaemolyticus on stored fish (Labeo rohita). When contaminated fish were kept at 0° to 4°C and at ?20°C the number of Vibrio parahaemolyticus was reduced significantly at ?20°C. After heat treatment (65, 70, 75 and 80°C for 1 min) no Vibrio parahaemolyticus was found on fish subjected to 80°C. Ultra-violet radiation resulted in considerable reductions in numbers of Vibrio parahaemolyticus after exposure for 15 min and still greater reductions after 30 min.     

Effects of temperature and photoperiod on the conditioning of the flat oyster (Ostrea edulis L.) in autumn

The production of the flat oyster Ostrea edulis (L.) natural spat in Europe has decreased almost by 60% in the past ten years. Thus, the importance of the production of oyster spat in hatcheries is evident. One of the critical steps in hatchery production is broodstock conditioning, especially difficult in autumn, when gonadal development is in resting period. Conditioning is influence by temperature, photoperiod and nutrition. In this work, the effects of two temperature and three photoperiod regimes on the conditioning of O. edulis were studied for three years by stereological analyses and registering number and dates of spawning and larval yield. Temperature had a positive effect on the gonadal development of O. edulis during conditioning. The percentages of germinal cells in oysters conditioned with a gradient of temperature (14–18°C) were double compared to oysters conditioned at 15°C. Oysters conditioned with longer photoperiods showed higher percentages of germinal cells. There was no interaction between temperature and photoperiod. Spawning was observed in the oysters treated with daylight (8–16 h) ten weeks from the beginning of conditioning. Flat oysters conditioned with 8 h and 8–12 h of daylight delayed the first spawning for a month. Total larval production was higher in the oysters treated with the longest daylight gradient. Gonadal and gametogenic development was a non‐synchronic process and the spawning extended for around two months. A protocol for flat oyster broodstock conditioning in autumn by using both a gradient of temperature (14–18°C) and daylight (8–16 h) is proposed.     

Effects of Ozonated Water Treatment on the Microbial Population,Quality, and Shelf Life of Shucked Oysters (Crassostrea plicatula)

An ozonated water treatment system was designed for the sanitation of shucked oysters (Crassostrea plicatula). The effects of ozone (O₃) on shelf life were examined under refrigerated conditions (4 ± 1°C). Microbiological, chemical, and color quality were also evaluated by determining total aerobic plate counts (APC), total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA), and L, a, and b values. Single and orthogonal experiment results showed that oysters treated with 9 mg/L of O₃ in aqueous solution (oyster: ozonated water = 1:6, w/v) for 10 min at 5°C significantly reduced the total APC by about 2-log—from 4.97 ± 0.11 to 2.93 ± 0.17 log CFU/g (p < 0.05). O₃ treatment did not cause significant changes in TVB-N, TBA, a, and b values, although L values were significantly affected. During refrigerated storage, O₃-treated samples expressed lower TVB-N, TBA, a, and b values and higher L values than control samples. The shelf life of O₃-treated samples was 20 to 25 days, while that for the controls was 5 to 10 days. The current study confirms that treatment with ozonated water can effectively prolong the shelf life and maintain the quality of oysters.     

Prevalence and serotyping of Listeria monocytogenes in Portuguese live bivalve molluscs sampled in various steps along the sanitary control process

The prevalence and contamination levels of Listeria monocytogenes were investigated in live bivalve molluscs for human consumption, collected in various steps of the commercial and sanitary circuits in the North of Portugal. Samples of different species were collected per lot before and after depuration treatment in two depuration units and further, when placed in retail markets. Listeria monocytogenes was isolated from 4% of the samples although with very low contamination levels (less than 100 MPN g^?1). None of the environmental (non‐depurated) samples were contaminated with the food‐borne pathogen. The positive samples involved depurated and commercialized samples from two lots, one in each circuit. Two serotypes (1/2a and 3b) were detected among the isolated strains. This study demonstrated the possibility of occurrence of L. monocytogenes contamination of live bivalve molluscs along the sanitary control circuit, including at the retail market level. As L. monocytogenes grows well at refrigerated temperatures and in high salt matrixes such as seafood its presence in these products should not be overlooked.     

Seasonal Fouling Stress on the Farmed Pearl Oyster,Pinctada fucata,from Southeastern Arabian Sea

Biofouling on the periostracum of pearl oysters and on the cages has been considered as a stress factor causing mortality of the farmed stock, reducing growth rates and also affecting pearl quality. In farming experiments using the pearl oyster, Pinctada fucata, at Kollam Bay (India), biofouling was found to be a problem. This study was conducted to understand the effect of fouling on the mortality of pearl oysters kept in suspended culture, to identify the main foulers, the seasonal variation in biofouling and species successions in the community with reference to abiotic factors. The average monthly mortality rate (MR) was estimated as 0.117 ± 0.002 and the monthly variations were significantly different (P < 0.01). The total fouling (0.163 ± 0.002 g/g oyster) and biofouling weights (0.166 ± 0.007 g/g oyster) were high in December when the fouling community was composed of several species and dominated by the ascidian, Didemnum sp. December was also the period when the MRs peaked indicating that this organism was the main cause of mortality in pearl farms in Kollam Bay. A clear seasonality in the fouling community (25 species belonging to nine phyla) has been observed in the present study. On the basis of this study, monthly cleaning of oysters is advocated except during December, January, and March when the cleaning should be fortnightly.     

Molecular cloning and expression of the heat shock protein 70 gene in the Kumamoto oyster <Emphasis Type="Italic">Crassostrea sikamea</Emphasis>

Episodes of summer mortality of the Kumamoto oyster Crassostrea sikamea are a major problem for its cultivation. Expression of the heat shock protein 70 (HSP70) is induced by various environmental stresses, including heat. We cloned and sequenced hsp70 complementary DNA from C. sikamea to investigate the relationship between hsp70 expression and heat tolerance in this oyster. Quantitative real-time polymerase chain reaction was performed using gill tissue dissected from oysters before and after heat shock for 1 h. The results showed hsp70 expression was faster and greater in oysters cultured at 20–22 °C than at 10–12 °C, and survival was lower among oysters cultured at 20–22 °C than at 10–12 °C. Moreover, heat tolerance was investigated by a 1-h pre-heat treatment, followed by exposure to heat shock conditions 5 days later. Survival was higher and hsp70 expression was notably lower in oysters that received the pre-heat treatment compared with those that did not. We conclude that a pre-heat treatment of only 1 h may be useful for inducing heat tolerance in C. sikamea, and that a low level of hsp70 expression after heat shock is an important index in selecting for high heat tolerance in these oysters.     

Growth and gonad development of the tropical black-lip pearl oyster, Pinctada margaritifera (L.), in the Gambier archipelago (French Polynesia)

The growth and reproductive cycle of cultured black-lipped pearl oysters, Pinctada margaritifera (L.), were studied in the Gambier Islands (134°52′ W, 23°07′ S) from September 2002 to August 2003. Temperatures were recorded throughout the year, revealing seasonal temperature variations between 22.3 and 27.8°C. The mean annual chlorophyll a value, as computed from satellite data, was 0.188 ± 0.075 μg L⁻¹. To study growth and reproduction, 720 two-year-old individuals were ear hung on long-lines suspended at a depth of 7 m. Samples were taken twice a month to obtain the following measurements: shell height; wet weight of flesh and total oyster; dry weight of adductor muscle, mantle and visceral mass; and glycogen content. Gonad development was also studied by histology on parallel samples. Growth was relatively fast during the first 6 months of the study: average shell height increased from 89.1 ± 9.1 to 119.7 ± 10.8 mm and total weight from 93.4 ± 24.5 to 155.1 ± 33.6 g, between September and the end of March. Subsequently, from April to August, no significant growth was observed for shell and flesh, while the muscle weight decreased significantly. Condition index (CI), defined as the ratio of wet weight of the visceral mass to shell weight, and histological changes in the gonad revealed 3 significant reproductive events of different intensities. The analysis of correlations revealed a specific effect of the chlorophyll a concentration on the growth of shell and soma, and one of the temperature on tissue glycogen content. This study also showed also that CI could be an efficient indicator of reproductive events in pearl oyster. It thus appears that the development of gonads goes on throughout the year in the Gambier Islands, without any detectable phase of sexual rest.