Control Oysters Research Articles

Costs of induced defenses dissipate by maturity for diploid and triploid oysters

Oyster reef restoration efforts and on-bottom aquaculture are frequently plagued with high predation rates. Oysters are phenotypically plastic, and rearing juvenile oysters, Crassostrea virginica, with predator cues causes them to grow stronger shells that increases survivorship in the field. However, induced defenses (e.g., shell hardening in oysters) are often associated with cost-benefit trade-offs, and the extent the increased shell strength persists into adulthood and alters the growth of somatic and reproductive tissues remains unknown. We raised diploid oysters (used in reef restoration) and triploid oysters (used in aquaculture) with and without predator cues for one month before placing individuals on an oyster farm to grow to market size. Oyster shell characteristics, soft tissue mass, and reproductive investment were measured periodically over one year of culture and compared across treatments. Both diploid and triploid oysters had significantly stronger and smaller shells than controls at the end of their nursery period. However, while diploid shells became 15 % stronger and 17 % smaller than controls, triploid shells became 28 % stronger and 23 % smaller. Additionally, triploid oysters exposed to predator cues returned to the size of controls faster and maintained their shell strength differences longer than diploids. Differences in soft tissue mass between treatments mirrored the patterns exhibited in shell size and weight with greater initial physiological costs and faster recovery for triploid individuals. There was no significant difference in somatic or reproductive tissue mass between induced and control oysters of the same ploidy after seven months in the field. Triploid oysters were 15–110 % larger than diploids depending on the characteristic measured at maturity. Additionally, there was a significant interaction between treatment and ploidy because induced triploids had marginally greater growth than their control counterparts while induced diploids had marginally less growth than controls. These findings demonstrate that physiological costs of oysters reacting to predators in early life stages are minimal by the time individuals reach maturity. Early exposure to predator cues is a promising tool for improving oyster survivorship in restoration and aquaculture operations, especially in regions with high predation pressure.

Evidence for immune priming specificity and cross-protection against sympatric and allopatric Vibrio splendidus strains in the oyster Magalana (Crassostrea) gigas

Infections with pathogenic Vibrio strains are associated with high summer mortalities of Pacific oysters Magalana (Crassostrea) gigas, affecting production worldwide. This raises the question of how M. gigas cultures can be protected against deadly Vibro infection. There is increasing experimental evidence of immune priming in invertebrates, where previous exposure to a low pathogen load boosts the immune response upon secondary exposure. Priming responses, however, appear to vary in their specificity across host and parasite taxa. To test priming specificity in the Vibrio – M. gigas system, we used two closely related Vibrio splendidus strains with differing degrees of virulence towards M. gigas. These V. splendidus strains were either isolated in the same location as the oysters (sympatric, opening up the potential for co-evolution) or in a different location (allopatric). We extracted cell-free haemolymph plasma from infected and control oysters to test the influence of humoral immune effectors on bacterial growth in vitro. While addition of haemolypmph plasma in general promoted growth of both strains, priming by an exposure to a sublethal dose of bacterial cells lead to inhibitory effects against a subsequent challenge with a potentially lethal dose in vitro. Inhibitory effects and immune priming was strongest when oysters had been primed with the sympatric Vibrio strain, but inhibitory effects were seen both when challenged with the sympatric as well as against allopatric V. splendidus, suggesting some degree of cross protection. The stronger immune priming against the sympatric strain suggests that priming could be more efficient against matching local strains potentially adding a component of local adaptation or co-evolution to immune priming in oysters. These in vitro results, however, were not reflected in the in vivo infection data, where we saw increased bacterial loads following an initial challenge. This discrepancy might suggests that that it is the humoral part of the oyster immune system that produces the priming effects seen in our in vitro experiments.

Effect of Different Cold Storage Temperatures on the Evolution of Shucking Yield and Quality Properties of Offshore Cultured Japanese Oyster (Magallana gigas) Treated by High Pressure Processing (HPP)

High pressure processing (HPP) can improve oyster shucking yield immediately after the treatment and increase the microbiological and sensory shelf life of oysters stored at 0-4 °C. However, the evolution of shucking yield during storage has not been previously examined and there are no studies focusing on shelf life at higher storage temperatures. To elucidate both aspects, control and HPP (300 MPa; 2 min) offshore cultivated oysters (Magallana gigas) were stored at 4 and 10 °C for 14 days, analyzing shucking yield, color, texture, microbiological and sensory characteristics. HPP samples showed a higher shucking yield (17% on average) than controls with minimal impact in texture and color, regardless of storage time and temperature. At 10 °C, HPP delayed microbial growth and sensory deterioration, increasing the estimated shelf life of oysters by 3 to 4 days (aerobic plate count < 6 log cycles; overall sensory acceptability > 2). Compared to controls stored at 4 °C, HPP oysters stored at 10 °C presented the same shelf life (5 to 9 days) but with higher shucking yield (up to 25%). In conclusion, HPP is an excellent tool to increase the shucking yield and delay sensory deterioration of oysters stored at 10 °C.

Legacy of past exposure to hypoxia and warming regulates an ecosystem service provided by oysters.

Climate change is having substantial impacts on organism fitness and ability to deliver critical ecosystem services, but these effects are often examined only in response to current environments. Past exposure to stress can also affect individuals via carryover effects, and whether these effects scale from individuals to influence ecosystem function and services is unknown. We explored within-generation carryover effects of two coastal climate change stressors-hypoxia and warming-on oyster (Crassostrea virginica) growth and nitrogen bioassimilation, an important ecosystem service. Oysters were exposed to a factorial combination of two temperature and two diel-cycling dissolved oxygen treatments at 3-months-old and again 1 year later. Carryover effects of hypoxia and warming influenced oyster growth and nitrogen storage in complex and context-dependent ways. When operating, carryover effects of single stressors generally reduced oyster nitrogen bioassimilation and relative investment in tissue versus shell growth, particularly in warm environments, while early life exposure to multiple stressors generally allowed oysters to perform as well as control oysters. When extrapolated to the reef scale, carryover effects decreased nitrogen stored by modeled oyster reefs in most conditions, with reductions as large as 41%, a substantial decline in a critical ecosystem service. In some scenarios, however, carryover effects increased nitrogen storage by modeled oyster reefs, again highlighting the complexity of these effects. Hence, even brief exposure to climate change stressors early in life may have persistent effects on an ecosystem service 1 year later. Our results show for the first time that within-generation carryover effects on individual phenotypes can impact processes at the ecosystem scale and may therefore be an overlooked factor determining ecosystem service delivery in response to anthropogenic change.

Effects of Farm Location on Vibrio parahaemolyticus and Vibrio vulnificus Levels in Oysters after Desiccation and Resubmersion in the Northern Gulf of Mexico

Desiccation is a routine farming practice used in off-bottom oyster aquaculture to reduce biofouling organisms and improve shell quality. This practice can increase Vibrio parahaemolyticus and Vibrio vulnificus levels, leading to increased risk of illness for raw oyster consumers. Previous resubmersion studies were performed in geographic proximity to one another, so to better understand the broader applicability of resubmersion, the next step was to perform concurrent studies in multiple geographic locations within a region. This study evaluated the effect of variations in geographic location on the recovery time needed for elevated vibrio levels to return to ambient levels in desiccated oysters after resubmersion at Gulf Coast farms. Two trials were performed between May and August 2019 at sites spanning ∼100 km: three in Alabama and one in Florida. Oysters were deployed in OysterGro cages at each location, 2 weeks before each trial, and then either were desiccated for 24 h or remained submersed as controls. Triplicate samples were taken before and immediately following the desiccation period, as well as 7 and 14 days after resubmersion. Total and pathogenic V. parahaemolyticus and V. vulnificus levels were determined using most-probable-number (MPN) real-time PCR. Vibrio levels increased by 0.23 to 3.50 log MPN/g after desiccation. Recovery times varied among geographic locations by trial and Vibrio spp., with all vibrio counts recovering to levels not significantly higher than those in control oysters within 7 to 14 days of resubmersion (P ≥ 0.06). These results suggest a 14-day resubmersion period of cultured oysters allowed vibrio levels, elevated because of routine handling, to return to ambient levels at all farm sites studied.

Electrophysiological Evaluation of Pacific Oyster (Crassostrea gigas) Sensitivity to Saxitoxin and Tetrodotoxin.

Pacific oysters (Crassostrea gigas) may bio-accumulate high levels of paralytic shellfish toxins (PST) during harmful algal blooms of the genus Alexandrium. These blooms regularly occur in coastal waters, affecting oyster health and marketability. The aim of our study was to analyse the PST-sensitivity of nerves of Pacific oysters in relation with toxin bio-accumulation. The results show that C. gigas nerves have micromolar range of saxitoxin (STX) sensitivity, thus providing intermediate STX sensitivity compared to other bivalve species. However, theses nerves were much less sensitive to tetrodotoxin. The STX-sensitivity of compound nerve action potential (CNAP) recorded from oysters experimentally fed with Alexandrium minutum (toxic-alga-exposed oysters), or Tisochrysis lutea, a non-toxic microalga (control oysters), revealed that oysters could be separated into STX-resistant and STX-sensitive categories, regardless of the diet. Moreover, the percentage of toxin-sensitive nerves was lower, and the STX concentration necessary to inhibit 50% of CNAP higher, in recently toxic-alga-exposed oysters than in control bivalves. However, no obvious correlation was observed between nerve sensitivity to STX and the STX content in oyster digestive glands. None of the nerves isolated from wild and farmed oysters was detected to be sensitive to tetrodotoxin. In conclusion, this study highlights the good potential of cerebrovisceral nerves of Pacific oysters for electrophysiological and pharmacological studies. In addition, this study shows, for the first time, that C. gigas nerves have micromolar range of STX sensitivity. The STX sensitivity decreases, at least temporary, upon recent oyster exposure to dinoflagellates producing PST under natural, but not experimental environment.

Effect of Gear Type on Vibrio spp. Levels in Farm-Raised Oysters (Crassostrea virginica) after Routine Handling and Resubmersion

During routine handling, cultured oysters are removed from the water and exposed to elevated temperatures, causing growth of Vibrio vulnificus and Vibrio parahaemolyticus within them. Farmers can resubmerse oysters in the water, allowing elevated Vibrio spp. levels to return to ambient levels within the oysters. Previous resubmersion research is limited to one aquaculture gear type during studies performed from June to September. This study aims to expand knowledge about the recovery times needed for elevated Vibrio levels in handled oysters from two common gear types (the adjustable longline system and the OysterGro system) during early and midsummer periods. Oysters held in both gear types were subjected to being tumbled and refrigerated or desiccated and then resubmersed into water in May and July 2018 and 2019. Vibrio spp. levels were measured before and after the treatments, and 3, 7, and 14 days after resubmersion, and were compared with levels in submersed oysters. All samples were tested for V. vulnificus, total V. parahaemolyticus, and pathogenic V. parahaemolyticus (tdh+/trh+). Water temperatures in May were significantly lower (∼5°C; P ≤ 0.009) than in July, corresponding to lower V. vulnificus levels (-0.67 log MPN/g) and higher tdh+/trh+ levels (+0.56 to 0.63 log MPN/g) in control oysters. The average Vibrio spp. levels in control oysters from each trial did not differ between the gear types (P ≥ 0.08). Elevated V. vulnificus levels recovered to ambient levels after 7 days in May and 3 days in July, regardless of gear or handling. For V. parahaemolyticus, the desiccated oysters required 14 days to recover in May and 7 days in July, whereas the tumbled and refrigerated oysters required 14 days or more in both months. This study had limited replication in each month, but the data suggest that the resubmersion times differ between the gear types, treatment types, and months. Future studies with more replications are needed to determine whether these trends continue.

Effects of tumbling, refrigeration and subsequent resubmersion on the abundance of Vibrio vulnificus and Vibrio parahaemolyticus in cultured oysters (Crassostrea virginica)

Routine handling of oysters is a common industry practice for off-bottom oyster aquaculture, which aims to produce a high-quality oyster. These practices expose oysters to elevated temperatures and interrupt filter feeding, which can increase Vibrio vulnificus and V. parahaemolyticus levels within the oyster. The resubmersion of oysters after exposure to conditions where the time-temperature controls are exceeded is as an effective mitigation strategy to allow elevated levels of Vibrio spp. to “recover”, or return to ambient levels, prior to harvest. Previous work examined the effect of desiccation on recovery times; the objective of this study was to evaluate the effect of additional handling treatments [tumbled and refrigerated (TR), tumbled and not refrigerated (TNR), not tumbled and refrigerated (NTR), and not tumbled and not refrigerated (NTNR)] on the time needed for V. vulnificus, total V. parahaemolyticus, and pathogenic V. parahaemolyticus (tdh+/trh+) to recover in oysters. A set of non-treated (control) oysters remained submerged throughout the study to determine the ambient Vibrio spp. (inclusive of genotypes) levels within oysters. Vibrio spp. levels were measured immediately before (pre) and after (post) the treatments, and 1, 2, 4, 7, 10, and 14 days after resubmersion using a three-tube MPN real-time PCR method. The non-refrigerated oysters (TNR, NTNR) had Vibrio spp. levels 1.54 to 2.10 log MPN/g higher than the pre-treatment levels, while the Vibrio spp. levels in refrigerated oysters were not significantly higher than pre-treatment levels. After resubmersion, Vibrio spp. levels increased by 0.84 to 1.78 log MPN/g in the refrigerated oysters (TR, NTR). Vibrio spp. levels in oysters returned to ambient after 1–7 days of resubmersion, depending on the handling treatment and the Vibrio spp. These results provide data on handling treatments not previously reported and further support the seven-day resubmersion requirement for farmers in Alabama using the adjustable longline system.

Inoculation With Desulfovibrio sp. Does Not Enhance Chalk Formation in the Pacific Oyster

Some oyster species precipitate a soft, friable form of calcite that is occluded within their shells, often referred to as chalk or chalky deposits. Because of the unusual microstructure of this shell feature, it has been proposed that chalk is the result of microbial involvement in the calcification process. Specifically, chalk has been hypothesized to be induced or influenced by microbial sulfate-reduction, and therefore chalk formation may not be under direct control of the oysters themselves. Specimens of the Pacific oyster, Magallana gigas, well known for chalk deposition within shells, were grown in Bodega Harbor, Bodega Bay, California and exposed monthly to treatments that altered the abundance of sulfate-reducing bacteria within oysters. The four treatment conditions evaluated included a control group (not exposed to any reagents), as well as oysters exposed to ciprofloxacin (a broad spectrum antibiotic), sodium molybdate (a compound known to inhibit bacterial sulfate reduction), and an inoculum of a sulfate-reducing bacterium isolated for this study. At the end of a 7 month growing period, specimens were culled and shells from treatment groups were assessed for chalk content by measuring bulk shell density and percentage of chalk in a cross sectional area. While analyses show that treatment conditions were successful with respect to altering abundances of sulfate-reducing bacteria in oysters, increasing SRB populations did not correlate to enhanced chalk expression in oyster shells. Therefore, chalk formation in oysters is not induced by the activity of sulfate-reducing bacteria within the oyster. Interestingly, control oysters produced more chalk than the other treatment groups, according to both bulk shell density and percent chalk measured in cross section. Given that control oysters represent the wild type for chalk expression in shells, it is inferred that the decreased formation of chalk in the other groups was due to a perturbation of the microbiome in the oyster calcifying fluid. However, the methods used here only quantify the presence of sulfate-reducing bacteria in oysters, and therefore, additional work is necessary to evaluate the role of the microbiome in oyster calcification.

Testing the efficacy of bouncing-bucket nursery systems for enhancing shell strength and thickness in on-bottom cultured Eastern oysters (Crassostrea virginica)

Through physical manipulation, oyster growers can modify the morphological features of young oysters in order to improve their robustness and resiliency to predation. In this study, the efficacy of a novel passive culturing technique using floating buckets that “bounce” oysters prior to the benthic grow-out phase for enhancing shell thickness and strength, while not compromising oyster shape, was investigated. Using a field experiment, we quantified and compared the shell thickness (mm), compressive strength (Newtons), and shape (fan ratios and cup ratios) of juvenile oysters subjected to this bouncing technique with oysters grown in floating bags, the current industry standard. Results indicated that bouncing increased shell thickness and strength for oysters >45 mm shell height compared to control oysters, but also decreased shell thickness and strength for oysters <45 mm shell height compared to control oysters. There was no difference in shell shape between bounced and control oysters, with both groups having similar fan and cup ratios. Ultimately, our results suggest that the size at which bounced oysters are released to the benthos for grow out will dictate the usefulness of bouncing technique for reducing predation-related mortality.

Unraveling concordant and varying responses of oyster species to Ostreid Herpesvirus 1 variants

The Ostreid herpesvirus 1 (OsHV-1) and variants, particularly the microvariants (μVars), are virulent and economically devastating viruses impacting oysters. Since 2008 OsHV-1 μVars have emerged rapidly having particularly damaging effects on aquaculture industries in Europe, Australia and New Zealand. We conducted field trials in Tomales Bay (TB), California where a non-μVar strain of OsHV-1 is established and demonstrated differential mortality of naturally exposed seed of three stocks of Pacific oyster, Crassostrea gigas, and one stock of Kumamoto oyster, C. sikamea. Oysters exposed in the field experienced differential mortality that ranged from 64 to 99% in Pacific oysters (Tasmania>Midori = Willapa stocks), which was much higher than that of Kumamoto oysters (25%). Injection trials were done using French (FRA) and Australian (AUS) μVars with the same oyster stocks as planted in the field and, in addition, two stocks of the Eastern oyster, C. virginica. No mortality was observed in control oysters. One C. virginica stock suffered ~10% mortality when challenged with both μVars tested. Two Pacific oyster stocks suffered 75 to 90% mortality, while one C. gigas stock had relatively low mortality when challenged with the AUS μVar (~22%) and higher mortality when challenged with the French μVar (~72%). Conversely, C. sikamea suffered lower mortality when challenged with the French μVar (~22%) and higher mortality with the AUS μVar (~44%). All dead oysters had higher viral loads (~1000×) as measured by quantitative PCR relative to those that survived. However, some survivors had high levels of virus, including those from species with lower mortality. Field mortality in TB correlated with laboratory mortality of the FRA μVar (69% correlation) but not with that of the AUS μVar, which also lacked correlation with the FRA μVar. The variation in response to OsHV-1 variant challenges by oyster species and stocks demonstrates the need for empirical assessment of multiple OsHV-1 variants.

Influence of environment on the pathogenesis of Ostreid herpesvirus-1 (OsHV-1) infections in Pacific oysters (Crassostrea gigas) through differential microbiome responses

The oyster microbiome is thought to contribute to the pathogenesis of mass mortality disease in Pacific oysters, associated with OsHV-1. As filter-feeders, oysters host a microbiota that can be influenced by the estuarine environment. This may alter susceptibility to OsHV-1 infections, causing variable mortality. This study aimed at: (1) differences in the microbiome of Pacific oysters with a common origin but grown in geographically distinct estuaries; (2) evaluating changes occurring in the microbiota, especially in Vibrio, and (3) differential responses of the oyster microbiome, in response to an OsHV-1 infection. Pacific oysters sourced from a single hatchery but raised separately in Patonga Creek, Shoalhaven River and Clyde River of NSW, Australia, were used and challenged with OsHV-1. The initial microbiome composition was different in the three batches and changed further, post-injection (p < 0.05). The Patonga oysters with the highest mortality also had higher OsHV-1 and Vibrio quantities compared to the other two batches (p < 0.05). The higher initial bacterial diversity in Patonga oysters decreased in moribund oysters which was not observed in the other two batches (p < 0.05). The microbiome of survivors of OsHV-1 infection and negative control oysters of two batches, did not show any changes with the relevant pre-challenged microbiome. A strong correlation was observed between the OsHV-1 and Vibrio quantities in OsHV-1 infected oysters (r = 0.6; p < 0.001). In conclusion, the Pacific oyster microbiome differed in different batches despite a common hatchery origin. Different microbiomes responded differently with a differential outcome of OsHV-1 challenge. The higher Vibrio load in oysters with higher OsHV-1 content and higher mortality, suggests a role in Vibrio in the pathogenesis of this mortality disease. This study provided insights of the potential of different estuarine environments to shape the Pacific oyster microbiome and how different microbiomes are associated with different outcomes of OsHV-1 infection.

Oyster (Crassostrea gasar) gastrointestinal tract microbiota and immunological responses after antibiotic administration

Microbiota, the microorganisms that colonize living organisms. Oysters rely exclusively on an innate immune system. This study characterized the cultivable gastrointestinal tract microbiota (GTM) of the oyster Crassostrea gasar and evaluated their influence on immune responses. Antibiotics were used to reduce or alter the microbiota during in vitro and in vivo assays. Haemocyte viability, concentration, and phagocytic capacity, as well as the production of reactive oxygen species (ROS), were evaluated in antibiotic-treated and control oysters. Microbiological analysis of the gastrointestinal tract was also performed; bacteria were selected using culture media and were identified by 16S ribosomal DNA. The results showed that the antibiotics eliminated bacteria in vitro but increased their concentrations in vivo . The GTM was composed of 26% amylolytic bacteria, 21% lipolytic bacteria, 18% proteolytic bacteria, 18% cellulolytic bacteria and 17% lactic acid bacteria; there were no differences in the amounts of these bacterial types between the control and treated oysters. Three major bacterial phyla, Proteobacteria, Firmicutes and Actinobacteria, and seven genera, Labrenzia , Pseudomonas , Halomonas , Shewanella , Vibrio , Bacillus , and Micrococcus , were detected. The concentration, viability, and phagocytic capacity of hemocytes and the production of ROS were not significantly altered by antibiotic treatment. In conclusion, the antibiotics altered the number of heterotrophic bacteria without changing hemocyte function, suggesting that this assay could be useful for verifying the influence of microbiota in host-parasite interactions.

Improved Microbial Safety of Direct Ozone-Depurated Shellstock Eastern Oysters (Crassostrea virginica) by Superchilled Storage.

The effect of superchilled storage at -1°C on the microbial safety of oyster depurated with 0.2, 0.4, and 0.6 mg/L ozone was studied for 14 days. Fecal coliforms (4,100–16,000 MPN/100 g), Escherichia coli (1,500–3,650 MPN/100 g), Vibrio cholerae non-O1/non-O139 (13.0–102.0 MPN/g), and Salmonella spp. (2.270–3.035 × 103 CFU/g) were initially present in raw oysters. After 6 h depuration, fecal coliform counts decreased (P < 0.05) to 300, 20 and 20 MPN/100 g for 0.2, 0.4, and 0.6 mg/L treatments, while a 0.3 log decrease in control oysters was observed. Initial E. coli counts decreased (P < 0.05) in oysters to 50, 20, and 20 MPN/100 g for 0.2, 0.4, and 0.6 mg/L treatments, respectively. A 1 log reduction in V. cholerae non-O1/non-139 levels were observed in 0.4 and 0.6 mg/L-treatments after 2 and 4 h depuration. Salmonella spp. was not detected in oyster samples after 6 h depuration in 0.4 and 0.6 mg/L-ozone treatments. Considering the bacterial loads after depuration, at the end of superchilled storage the 0.4 mg/L-ozonated oysters attained lower (P < 0.05) fecal coliform levels (280 MPN/100 g) and E. coli counts in 0.4 and 0.6 mg/L-ozonated oysters (20 and 95 MPN/100 g, respectively). A 2-log decrease in V. cholerae non-O1/non-O139 levels on day 5 in 0.4 and 0.6 mg/L-ozonated oysters (< 0.3 MPN/g) was attained. V. cholerae non-O1/non-O139 counts in control oysters decreased 1 log on day 9 of superchilled storage. Salmonella spp. was not detected in ozonated and superchilled stored oysters. Levels of fecal coliforms, E. coli, Salmonella spp., and V. cholerae non-O1/non-O139 in non-ozone depurated oyster samples were higher than in control, 0.4 and 0.6 mg/L ozonated oyster samples during superchilled storage. The cumulative mortality rates after 14 days of storage for superchilled oysters (22.2%) was higher (P < 0.05) than 0.6 mg/L O3 (7.2%) and 0.4 mg/L O3 (5.8%) treatments, and control oysters (5.6%). pH values in control oysters decreased significantly (P < 0.05) throughout the storage period but not in oysters of both ozone treatments, indicating no detrimental effects on oyster survival. The results of this study suggest that superchilled storage enables ozonated shellstock oysters (0.4 mg/L-6 h) stored for 9 days to be safe human consumption.

Expressed exome capture sequencing: A method for cost-effective exome sequencing for all organisms.

Exome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require annotated genomic resources. Here, we present a method for creating cDNA probes from expressed mRNA, which are then used to enrich and capture genomic DNA for exon regions. This approach, called "EecSeq," eliminates the need for costly probe design and synthesis. We tested EecSeq in the eastern oyster, Crassostrea virginica, using a controlled exposure experiment. Four adult oysters were heat shocked at 36°C for 1hr along with four control oysters kept at 14°C. Stranded mRNA libraries were prepared for two individuals from each treatment and pooled. Half of the combined library was used for probe synthesis, and half was sequenced to evaluate capture efficiency. Genomic DNA was extracted from all individuals, enriched via captured probes, and sequenced directly. We found that EecSeq had an average capture sensitivity of 86.8% across all known exons and had over 99.4% sensitivity for exons with detectable levels of expression in the mRNA library. For all mapped reads, over 47.9% mapped to exons and 37.0% mapped to expressed targets, which is similar to previously published exon capture studies. EecSeq displayed relatively even coverage within exons (i.e., minor "edge effects") and even coverage across exon GC content. We discovered 5,951 SNPs with a minimum average coverage of 80×, with 3,508 SNPs appearing in exonic regions. We show that EecSeq provides comparable, if not superior, specificity and capture efficiency compared to costly, traditional methods.

Does relaxation as a sedative therapy previous to pearl production affect antioxidant response and cause oxidative damage in the winged pearl oyster Pteria sterna?

Relaxation as a sedative therapy before nucleus seeding is assumed to be stressful to some marine animals during its initial phase, before acting at the cellular level. The antioxidant activity and possible oxidative damage to soft tissues was evaluated in the winged pearl oyster Pteria sterna exposed to four relaxants, at two concentrations each. The relaxants were: propylene phenoxetol (1.5 and 2.5 mL L−1), benzocaine (0.15 and 0.25 g L−1), magnesium chloride (15 and 30 g L−1), and eugenol (0.25 and 0.5 mL L−1). The enzymatic activity (superoxide dismutase, catalase, glutathione peroxidase) and oxidative damage (TBARS, lipofuscins) were measured at 30, 60, and 120 min after immersion in each relaxant, and at 65 h representing the recovery period (filtered seawater with no relaxant). Propylene phenoxetol (2.5 mL L−1) and benzocaine (0.25 g L−1) induced >80% relaxation within the first 45 min. Variations in the activity of superoxide dismutase activity were not significant between treatments in mantle and gills, but increased in muscle at 65 h in oysters exposed to 1.5 mL L−1 propylene phenoxetol and 15 and 30 g L−1 magnesium chloride. Control oysters significantly increased catalase activity in adductor muscle and gills (30–60 min), but the activity of glutathione peroxidase did not significant vary in muscle and mantle, and only decreased in gills at 65 h exposure to 2.5 mL L−1 propylene phenoxetol. Higher TBARS concentrations, as indicative of oxidative damage, only occurred at 30 min in muscle and gills in oysters exposed to 30 g L−1 magnesium chloride and 0.25 g L−1 benzocaine. Relaxing with benzocaine caused a significant accumulation of lipofuscins in gills at 120 min, again as possible indicator of oxidative stress; lowest lipofuscins values occurred with 1.5 mL L−1 propylene phenoxetol. These results confirm that propylene phenoxetol causes minimal interaction with antioxidant enzymes and provoke little oxidative damage to soft tissues in P. sterna.

Characterization of microbial community in high-pressure treated oysters by high-throughput sequencing technology

Abstract High-pressure (HP) processing technology has great application potential in bivalve mollusk industry. In this study, the effects of HP treatment on shucking and shelf-life extension of Pacific oysters (Crassostrea gigas) during 4 ± 1 °C storage were studied. Culture-independent high-throughput sequencing (HTS) technology was used to investigate the microbiota of oysters treated by HP (300 MPa, 2 min) during storage. Results showed that HP treatment at 275 MPa for 3 min or 300 MPa for 2 min could achieve 100% full release of oyster adductor muscle, pressures higher than 300 MPa caused disadvantageous damage of shells. Therefore, HP treatment at 300 MPa for 2 min was particularly appropriate. Based on organoleptic, biochemical and microbiological analyses, shelf-life of 6–8 days for control and 12 days for HP-treated oysters could be expected. The dominant bacteria in fresh oysters were Vibrio (28.3%), Shewanella (10.3%) and Pseudoalteromonas (7.2%). HP treatment changed the spoilage microbiota of oysters dramatically. Psychrobacter was dominant in the HP-treated spoiled oysters and its proportion was 42.3%, while Pseudoalteromonas (32.2%) and Shewanella (19.5%) were dominant in the spoiled oysters without HP treatment. This study confirmed that HP processing was effective in facilitating the removal of oyster meats from the shells and prolonging their shelf-life. More importantly, HP treatment changed the microbiota of oysters during chilled storage. Industrial relevance As seafood, oysters have a short shelf-life. High-pressure (HP) processing technology has showed great application potentials in the bivalve mollusk industry. In this work, we determined the effects of HP treatment on the shucking and shelf-life extension of oysters. We used high-throughput sequencing technology to investigate the microbiota of control and HP-treated oysters during refrigerated storage. We found that HP treatment changed the spoilage microbiota of oysters dramatically. Psychrobacter was dominant in the HP-treated spoiled oysters, while Pseudoalteromonas and Shewanella were dominant in the spoiled oysters without HP treatment. Therefore, controlling the growth of Psychrobacter could help to improve the quality of oysters treated by HP during storage.

Alterations of tissue metallothionein and vitellogenin concentrations in tropical cup oysters (Saccostrea sp.) following short-term (96 h) exposure to cadmium

Metallothioneins and vitellogenins are low molecular weight proteins that have been used widely in environmental monitoring as biomarkers of exposure and damage to metals and estrogenic compounds, respectively. In the present study, the responses of metallothionein and vitellogenin tissue concentrations were measured following acute (96h) aqueous exposures to cadmium in Saccostrea sp., a tropical cup oyster native to the Western Pacific Ocean that has recently established itself in the Caribbean Sea. Adult oysters (1.5–5.0cm shell length) collected from the municipal marina of Santa Marta, Colombia (Caribbean Sea) and acclimated for 5days in the laboratory, were exposed to Cd at five concentrations (0, 1, 10, 100 and 1000μg/L) and their tissues (gills, digestive gland and adductor muscle) were analyzed in pools of 5 individuals (3 replicates per concentration). Metallothioneins in digestive glands of oysters exposed to Cd concentrations≥100μg/L showed a significant increase, from 8.0 to 14.8μg MT/mg total protein, whereas metallothionein concentrations in gills increased to lesser extent, and no differences were observed in adductor muscle. Metallothionein concentrations in digestive gland and gills correlated directly with whole soft tissue Cd concentrations (ranging from 2 to 297μg/g dw Cd). Vitellogenin in homogenates of oyster gonad tissue, after 96h of exposure to 1000μg/L Cd, were significantly lower (0.04mg P/g gonad) compared to control oysters (0.68mg P/g gonad), suggestive of an anti-estrogenic effect of Cd at high concentrations, whereas no significant changes in vitellogenin concentrations were observed at intermediate Cd exposure concentrations. This study confirms acute responses of metallothionein and vitellogenin concentrations in tissues of Saccostrea sp. exposed to high concentrations of cadmium (Cd≥100μg/L, 96h). The present results are first step towards validating the use of these two proteins as biomarkers of metal exposure in this species.

Imidazole‐dicarboxylic Acid Nickel Complex as a Novel Grafting Agent for Pearl Oyster, Pinctada martensii

AbstractIn the pearl cultivation industry, the implantation of the nucleus is a critical step in the production process. The surgical insertion of the nucleus and the mantle results in a wound and stress that affect not only the survival chances of the pearl oysters but also the quality of the pearls produced. This study proposes a synthesized imidazole‐dicarboxylic acid nickel complex that may hold potential in assisting the wound healing and nacre formation processes. The acute toxicity of the complex to pearl oysters, Pinctada martensii, was examined in a dose–response study over a 90‐h exposure. The safe concentrations of the nickel complex determined using probit analysis were 0.916 mg/L and 0.471 mg/L for 72‐h and 96‐h treatments, respectively. The reliability of the regression was verified using the chi‐square test. The alkaline phosphatase (ALP) activity in different regions of the treated and control oysters, along with relevant blood biochemical indices, were measured over a 90‐d period to predict the potential effects of the nickel complex on wound healing and nacre formation. A significant increase in ALP activity in the mantle, together with elevated blood levels of Mg (II) and globulin, and reduced blood levels of Zn (II) and blood urea nitrogen were observed. These results indicate that the prepared nickel complex may hold promise in helping wound healing post mantle implantation, promote the secretion of nacre, and enhance the immunity of the oysters, thus ultimately improving the yield and quality of the pearls.

Effect of 4-nonylphenol on the immune response of the Pacific oyster Crassostrea gigas following bacterial infection with Vibrio campbellii

The xenoestrogen 4-nonylphenol (NP) is a ubiquitous aquatic pollutant and has been shown to impair reproduction, development, growth and, more recently, immune function in marine invertebrates. We investigated the effects of short-term (7 d) exposure to low (2 μg l−1) and high (100 μg l−1) levels of NP on cellular and humoral elements of the innate immune response of Crassostrea gigas to a bacterial challenge. To this end, we measured 1) total hemocyte counts (THC), 2) relative transcript abundance of ten immune-related genes (defh1, defh2, bigdef1, bigdef2, bpi, lysozyme-1, galectin, C-type lectin 2, timp, and transglutaminase) in the hemocytes, gill and mantle, and 3) hemolymph plasma lysozyme activity, following experimental Vibrio campbellii infection. Both low and high levels of NP were found to repress a bacteria-induced increase in THC observed in the control oysters. While several genes were differentially expressed following bacterial introduction (bigdef2, bpi, lysozyme-1, timp, transglutaminase), only two genes (bpi in the hemocytes, transglutaminase in the mantle) exhibited a different bacteria-induced expression profile following NP exposure, relative to the control oysters. Independently of infection-status, exposure to NP also altered mRNA transcript abundance of several genes (bpi, galectin, C-type lectin 2) in naïve, saline-injected oysters. Finally, plasma lysozyme activity levels were significantly higher in low dose NP-treated oysters (both naïve and bacteria challenged) relative to control oysters. Combined, these results suggest that exposure to ecologically-relevant (low) and extreme (high) levels of NP can alter both cellular and humoral elements of the innate immune response in C. gigas, an aquaculture species of global economic importance.