Toxicity of tributyltin to the European flat oyster Ostrea edulis: Metabolomic responses indicate impacts to energy metabolism, biochemical composition and reproductive maturation.

Tri-Butyl Tin (TBT) remains as a legacy pollutant in the benthic environments. Although the toxic impacts and endocrine disruption caused by TBT to gastropod molluscs have been established, the changes in energy reserves allocated to maintenance, growth, reproduction and survival of European oysters Ostrea edulis, a target species of concerted benthic habitat restoration projects, have not been explored. This study was designed to evaluate the effect of TBT chloride (TBTCl) on potential ions and relevant metabolomic pathways and its association with changes in physiological, biochemical and reproductive parameters in O. edulis exposed to environmental relevant concentrations of TBTCl. Oysters were exposed to TBTCl 20 ng/L (n = 30), 200 ng/L (n = 30) and 2000 ng/L (n = 30) for nine weeks. At the end of the exposure, gametogenic stage, sex, energy reserve content and metabolomic profiling analysis were conducted to elucidate the metabolic alterations that occur in individuals exposed to those compounds. Metabolite analysis showed significant changes in the digestive gland biochemistry in oysters exposed to TBTCl, decreasing tissue ATP concentrations through a combination of the disruption of the TCA cycle and other important molecular pathways involved in homeostasis, mitochondrial metabolism and antioxidant response. TBTCl exposure increased mortality and caused changes in the gametogenesis with cycle arrest in stages G0 and G1. Sex determination was affected by TBTCl exposure, increasing the proportion of oysters identified as males in O. edulis treated at 20ng/l TBTCl, and with an increased proportion of inactive stages in oysters treated with 2000 ng/l TBTCl. The presence and persistence of environmental pollutants, such as TBT, could represent an additional threat to the declining O. edulis populations and related taxa around the world, by increasing mortality, changing reproductive maturation, and disrupting metabolism. Our findings identify the need to consider additional factors (e.g. legacy pollution) when identifying coastal locations for shellfish restoration.

A small step or a giant leap: Accounting for settlement delay and dispersal in restoration planning.

Understanding larval duration and hence dispersal potential of the European oyster Ostrea edulis is crucial to inform restoration strategies. Laval duration has an obligatory period of maturity to pediveliger (when larvae are ready to settle), but also an unknown period until metamorphosis is triggered by a settlement cue. The extent to which larvae can prolong the pediveliger period and delay metamorphosis has not been studied. Here we show that O. edulis larvae can delay metamorphosis for a period of 11 days, while retaining the capability to settle in high proportions when presented with a suitable settlement cue. O. edulis larvae are likely to be able to delay metamorphosis even further, since 80% of larvae in the control treatment were still alive when the experiment was terminated at day 14. The results indicate the ability of O. edulis larvae to more than double pelagic duration and probably further delay metamorphosis. We discuss these findings in the context of larval mortality, and the importance of O. edulis' larval settlement requirements for dispersal potential, recruitment success and connectivity of restoration sites.

Conservation aquaculture as a tool for imperiled marine species: Evaluation of opportunities and risks for Olympia oysters, Ostrea lurida.

Conservation aquaculture is becoming an important tool to support the recovery of declining marine species and meet human needs. However, this tool comes with risks as well as rewards, which must be assessed to guide aquaculture activities and recovery efforts. Olympia oysters (Ostrea lurida) provide key ecosystem functions and services along the west coast of North America, but populations have declined to the point of local extinction in some estuaries. Here, we present a species-level, range-wide approach to strategically planning the use of aquaculture to promote recovery of Olympia oysters. We identified 12 benefits of culturing Olympia oysters, including identifying climate-resilient phenotypes that add diversity to growers' portfolios. We also identified 11 key risks, including potential negative ecological and genetic consequences associated with the transfer of hatchery-raised oysters into wild populations. Informed by these trade-offs, we identified ten priority estuaries where aquaculture is most likely to benefit Olympia oyster recovery. The two highest scoring estuaries have isolated populations with extreme recruitment limitation-issues that can be addressed via aquaculture if hatchery capacity is expanded in priority areas. By integrating social criteria, we evaluated which project types would likely meet the goals of local stakeholders in each estuary. Community restoration was most broadly suited to the priority areas, with limited commercial aquaculture and no current community harvest of the species, although this is a future stakeholder goal. The framework we developed to evaluate aquaculture as a tool to support species recovery is transferable to other systems and species globally; we provide a guide to prioritizing local knowledge and developing recommendations for implementation by using transparent criteria. Our collaborative process engaging diverse stakeholders including managers, scientists, Indigenous Tribal representatives, and shellfish growers can be used elsewhere to seek win-win opportunities to expand conservation aquaculture where benefits are maximized for both people and imperiled species.

Plastic response of the oyster Ostrea chilensis to temperature and pCO2 within the present natural range of variability.

Estuaries are characterized by high fluctuation of their environmental conditions. Environmental parameters measured show that the seawater properties of the Quempillén estuary (i.e. temperature, salinity, pCO2, pH and ΩCaCO3) were highly fluctuating and related with season and tide. We test the effects of increasing temperature and pCO2 in the seawater on the physiological energetics of the bivalve Ostrea chilensis. Juvenile oysters were exposed to an orthogonal combination of three temperatures (10, 15, and 20°C) and two pCO2 levels (~400 and ~1000 µatm) for a period of 60 days to evaluate the temporal effect (i.e. 10, 20, 30, 60 days) on the physiological rates of the oysters. Results indicated a significant effect of temperature and time of exposure on the clearance rate, while pCO2 and the interaction between pCO2 and the other factors studied did not show significant effects. Significant effects of temperature and time of exposure were also observed on the absorption rate, but not the pCO2 nor its interaction with other factors studied. Oxygen consumption was significantly affected by pCO2, temperature and time. Scope for growth was only significantly affected by time; despite this, the highest values were observed for individuals subject to to 20°C and to ~1000 µatm pCO2. In this study, Ostrea chilensis showed high phenotypic plasticity to respond to the high levels of temperature and pCO2 experienced in its habitat as no negative physiological effects were observed. Thus, the highly variable conditions of this organism's environment could select for individuals that are more resistant to future scenarios of climate change, mainly to warming and acidification.

Coming and going - Historical distributions of the European oyster Ostrea edulis Linnaeus, 1758 and the introduced slipper limpet Crepidula fornicata Linnaeus, 1758 in the North Sea.

Natural history collections are fundamental for biodiversity research as well as for any applied environment-related research. These collections can be seen as archives of earth´s life providing the basis to address highly relevant scientific questions such as how biodiversity changes in certain environments, either through evolutionary processes in a geological timescale, or by man-made transformation of habitats throughout the last decades and/or centuries. A prominent example is the decline of the European flat oyster Ostrea edulis Linneaus, 1758 in the North Sea and the concomitant invasion of the common limpet slipper Crepidula fornicata, which has been implicated to have negative effects on O. edulis. We used collections to analyse population changes in both species in the North Sea. In order to reconstruct the change in distribution and diversity over the past 200 years, we combined the temporal and spatial information recorded with the collected specimens contained in several European natural history collections. Our data recover the decline of O. edulis in the North Sea from the 19th century to the present and the process of invasion of C. fornicata. Importantly, the decline of O. edulis was nearly completed before C. fornicata appeared in the North Sea, suggesting that the latter had nothing to do with the local extinction of O. edulis in the North Sea.

Effects of the interaction between temperature and steroid hormones on gametogenesis and sex ratio in the European flat oyster (Ostrea edulis).

Throughout Europe, populations of Ostrea edulis have been in decline since the 1970s. Temperature has an important influence on physiological, biochemical and reproductive attributes of oysters. It is also the most easily modulated environmental factor in hatcheries, so it is useful to understand the implications of temperature variation in driving gametogenesis and sex development in a protandrous sequential hermaphrodites such as O. edulis. To understand the effect of temperature on gametogenesis and sex ratio, as well as the potential mechanism of sex determination through the role of steroid hormone homologues, oysters were exposed to three temperatures (10, 14, and 18 °C) for four months. Gametogenic stage and sex ratio were assessed histologically for each treatment. In parallel, concentrations of estradiol (E2)- and testosterone (T)- were determined in developing gonads. Our data show that by some biometric parameters, gametogenesis and sex ratio were significantly influenced by temperature during the experiment. There was a weak but significant correlation between E2 and T concentration during the treatments. However, and importantly, a direct relation between gonadal maturation, sex determination and hormones concentration was not found. These results suggest that gametogenesis and sex determination are predominantly affected by temperature in this species, and that steroids may not be actively involved as endogenous modulators in sex determination. Rising sea water temperatures and warmer condition through the year could cause an accelerated gametogenesis and skewed sex ratios in natural populations of O. edulis.

Conservation and restoration of a keystone species: Understanding the settlement preferences of the European oyster (Ostrea edulis).

The European oyster Ostrea edulis is a keystone species that is internationally recognised as 'threatened and declining' in the NE Atlantic by OSPAR and several nations have consequently adopted strategies for its conservation and restoration. Understanding the settlement behaviour of O. edulis larvae is crucial to inform these strategies. We compared the efficiency of several treatments in triggering settlement. The most effective settlement occurred with the presence of conspecifics: 100% settled in <23 h. Marine stones with habitat-associated biofilms induced 81% settlement that started after a 45 h delay. Sterile shells and terrestrial stones did not induce more settlement than control treatments. These results indicate that O. edulis larvae are gregarious and finely-tuned to settle in response to cues which are indicative of their adult habitat requirements. The role of chemical cues in mediating settlement, and the importance of this to restoration, are discussed.

Indications of future performance of native and non-native adult oysters under acidification and warming.

Globally, non-native species (NNS) have been introduced and now often entirely replace native species in captive aquaculture; in part, a result of a perceived greater resilience of NSS to climate change and disease. Here, the effects of ocean acidification and warming on metabolic rate, feeding rate, and somatic growth was assessed using two co-occurring species of oysters - the introduced Pacific oyster Magallana gigas (formerly Crassostrea gigas), and native flat oyster Ostrea edulis. Biological responses to increased temperature and pCO2 combinations were tested, the effects differing between species. Metabolic rates and energetic demands of both species were increased by warming but not by elevated pCO2. While acidification and warming did not affect the clearance rate of O. edulis, M. gigas displayed a 40% decrease at 750 ppm pCO2. Similarly, the condition index of O. edulis was unaffected, but that of M. gigas was negatively impacted by warming, likely due to increased energetic demands that were not compensated for by increased feeding. These findings suggest differing stress from anthropogenic CO2 emissions between species and contrary to expectations, this was higher in introduced M. gigas than in the native O. edulis. If these laboratory findings hold true for populations in the wild, then continued CO2 emissions can be expected to adversely affect the functioning and structure of M. gigas populations with significant ecological and economic repercussions, especially for aquaculture. Our findings strengthen arguments in favour of investment in O. edulis restoration in UK waters.

Effects of ocean acidification on 109Cd, 57Co, and 134Cs bioconcentration by the European oyster (Ostrea edulis): Biokinetics and tissue-to-subcellular partitioning.

The uptake and depuration kinetics of dissolved 109Cd, 57Co and 134Cs were determined experimentally in the European flat oyster Ostrea edulis (Linnaeus, 1758) under different pH conditions (i.e., 8.1, 7.8 and 7.5) for 59 days. Uptake and depuration rates were variable within these elements; no effects were observed under different pH conditions for the uptake biokinetics of 109Cd and 57Co and depuration of 109Cd and 134Cs in oyster. The uptake and depuration rate constants of 134Cs differed during the exposure phase between treatments, while the steady state concentration factors (CFss) were similar. The resulting Cs activity that was purged during short- and long-term depuration phases differed, while the remaining activities after thirty-nine days depuration phase (RA39d) were similar. Co-57 depuration was affected by pCO2 conditions: RA39d were found to be significantly higher in oysters reared in normocapnia (pCO2 = 350 µatm) compared to high pCO2 conditions. Co-57 tissue distribution did not differ among the variable pCO2 conditions, while 109Cd and 134Cs accumulated in soft tissue of oysters were found to be higher under the highest pCO2. Additionally, Cd, Co and Cs were stored differently in various compartments of the oyster cells, i.e. cellular debris, metal-rich granules (MRG) and metallothionein-like proteins (MTLP), respectively. The subcellular sequestration of the elements at the end of the depuration phase did not differ among pH treatments. These results suggest that bioconcentration and tissue/subcellular distribution are element-specific in the oyster, and the effects of higher pCO2 driven acidification and/or coastal acidification variably influence these processes.

Flat oyster follows the apoptosis pathway to defend against the protozoan parasite Bonamia ostreae.

The in vitro model Ostrea edulis hemocyte - Bonamia ostreae is interesting to investigate host-parasite interactions at the cellular level. Indeed, this unicellular parasite infects the flat oyster Ostrea edulis and multiplies within hemocytes, the central effectors of oyster defenses. Apoptosis is a mechanism used by many organisms to eliminate infected cells. In order to study the potential involvement of this mechanism in the oyster response to B. ostreae, in vitro experiments were carried out by exposing hemocytes from the naturally susceptible oyster O. edulis and a resistant oyster species Crassostrea gigas to live and heat-inactivated parasites. Hemocyte apoptotic response was measured using a combination of flow cytometry and microscopy analyses. Whatever the host species was, the parasite was engulfed in hemocytes and induced an increase of apoptotic parameters including intracytoplasmic calcium concentration, mitochondrial membrane potential or phosphatidyl-serine externalization as well as ultrastructural modifications. However, the parasite appears more able to infect flat oyster than cupped oyster hemocytes and the apoptotic response was more important against live than dead parasites in the natural host than in C. gigas. Our results suggest that O. edulis specifically responds to B. ostreae by inducing apoptosis of hemocytes.

Effects of microplastics on European flat oysters, Ostrea edulis and their associated benthic communities.

Plastic pollution is recognised as an emerging threat to aquatic ecosystems, with microplastics now the most abundant type of marine debris. Health effects caused by microplastics have been demonstrated at the species level, but impacts on ecological communities remain unknown. In this study, impacts of microplastics on the health and biological functioning of European flat oysters (Ostrea edulis) and on the structure of associated macrofaunal assemblages were assessed in an outdoor mesocosm experiment using intact sediment cores. Biodegradable and conventional microplastics were added at low (0.8 µg L(-1)) and high (80 µg L(-1)) doses in the water column repeatedly for 60 days. Effects on the oysters were minimal, but benthic assemblage structures differed and species richness and the total number of organisms were ∼1.2 and 1.5 times greater in control mesocosms than in those exposed to high doses of microplastics. Notably, abundances of juvenile Littorina sp. (periwinkles) and Idotea balthica (an isopod) were ∼2 and 8 times greater in controls than in mesocosms with the high dose of either type of microplastic. In addition, the biomass of Scrobicularia plana (peppery furrow shell clam) was ∼1.5 times greater in controls than in mesocosms with the high dose of microplastics. This work indicates that repeated exposure to high concentrations of microplastics could alter assemblages in an important marine habitat by reducing the abundance of benthic fauna.

Brooding strategy in fluctuating salinity environments: oxygen availability in the pallial cavity and metabolic stress in females and offspring in the Chilean oyster Ostrea chilensis.

Organisms that encounter stressful situations in nature often cope using behavioral (e.g., avoidance) or physiological tactics. In sessile mollusks, the only available behavioral option in dealing with salinity stress is to "clam up", isolating their tissues from the environment. Though effective in the short term, prolonged isolation can have detrimental physiological consequences, particularly for females brooding embryos in a mantle cavity that is isolated from the external environment. In the Quempillén estuary, the Chilean oyster, Ostrea chilensis, spent nearly one-third of its brooding season at salinities low enough to cause female isolation. When females thus isolated themselves, the dissolved oxygen in their mantle cavity fluid dropped to hypoxic levels within 10 min. In females that were brooding embryos, this depletion of oxygen was not uniform: oxygen was depleted more quickly in the palp region (where embryos accumulate) than in the inhalant region. Additionally, oxygen was reduced even more quickly in the palp region when females were brooding late-stage embryos, which consumed oxygen significantly more quickly than embryos in earlier developmental stages. Finally, O. chilensis used anaerobic metabolism to cope with the hypoxia induced by isolation, as lactate accumulated in the tissues of both females (brooding > non-brooding) and embryos (late stage > early stage). Our findings demonstrate the trade-off between an adaptive avoidance behavior (clamming up) and the potentially detrimental consequences brought on by such a behavior (hypoxia). Cycling of embryos throughout the mantle cavity by deliberate female pumping keeps them from accumulating in the area between the palps, forestalling the creation of hypoxic conditions there. In addition, the capacity for anaerobic metabolism by both females and their embryos should help them tolerate the low oxygen levels that do eventually arise when the pallial cavity is isolated from the surrounding environment during long periods of reduced ambient salinity.

Brooding in the Chilean oyster Ostrea chilensis: unexpected complexity in the movements of brooded offspring within the mantle cavity.

Brooding in invertebrates serves to protect embryos from stressful external conditions by retaining progeny inside the female body, effectively reducing the risk of pelagic stages being exposed to predation or other environmental stressors, but with accompanying changes in pallial fluid characteristics, including reduced oxygen availability. Brooded embryos are usually immobile and often encapsulated, but in some Ostrea species the embryos move freely inside the female pallial cavity in close association with the mother's gills for as long as eight weeks. We used endoscopic techniques to characterize the circulation pattern of embryos brooded by females of the oyster, Ostrea chilensis. Progeny at embryonic and veliger stages typically circulated in established patterns that included the use of dorsal and ventral food grooves (DFG, VFG) to move anteriorly on the gills. Both embryos and veligers accumulated around the mother's palps, and remained there until an active maternal countercurrent moved them to the gill inhalant area. Both food grooves were able to move embryos, veligers, and food-particle aggregates anteriorly, but the DFG was more important in progeny transport; early embryos were moved more rapidly than veligers in the DFG. A microcirculation pattern of embryos was apparent when they were moved by gill lamellae: when they were close to the VFG, most embryos lost gill contact and "fell" down to the DFG. Those that actually reached the DFG moved anteriorly, but others came into contact with the base of the lamellae and again moved towards the VFG. The circulation pattern of the progeny appears well-suited for both cleaning them and directing them posteriorly to an area where there is more oxygen and food than in the palp region. This process for actively circulating progeny involves the feeding structures (gill and palps) and appears to be energetically costly for the female. It also interferes with feeding, which could explain the poor energy balance previously documented for brooding females of this species.

Loss of an ecological baseline through the eradication of oyster reefs from coastal ecosystems and human memory.

Oyster reefs form over extensive areas and the diversity and productivity of sheltered coasts depend on them. Due to the relatively recent population growth of coastal settlements in Australia, we were able to evaluate the collapse and extirpation of native oyster reefs (Ostrea angasi) over the course of a commercial fishery. We used historical records to quantify commercial catch of O. angasi in southern Australia from early colonization, around 1836, to some of the last recorded catches in 1944 and used our estimates of catch and effort to map their past distribution and assess oyster abundance over 180 years. Significant declines in catch and effort occurred from 1886 to 1946 and no native oyster reefs occur today, but historically oyster reefs extended across more than 1,500 km of coastline. That oyster reefs were characteristic of much of the coastline of South Australia from 1836 to 1910 appears not to be known because there is no contemporary consideration of their ecological and economic value. Based on the concept of a shifted baseline, we consider this contemporary state to reflect a collective, intergenerational amnesia. Our model of generational amnesia accounts for differences in intergenerational expectations of food, economic value, and ecosystem services of nearshore areas. An ecological system that once surrounded much of the coast and possibly the past presence of oyster reefs altogether may be forgotten and could not only undermine progress towards their recovery, but also reduce our expectations of these coastal ecosystems.

Analysis of large-scale spatio-temporal trends of Ostrea puelchana beds in Northern Patagonian gulfs, Argentina.

Wild oyster populations have declined severely worldwide, however fluctuations of South Atlantic populations are poorly documented. We explored the changes in the abundance of Ostrea puelchana population of Northern Patagonia, Argentina, by linking data from paleontological, archaeological and informal sources, with time series data from fishing, ecological and studies of oyster pathology. The present work is the first study which includes a South Atlantic time series concerning oyster beds. The focal area for this study is the San Matías Gulf (SMG, 40° 50'-42° 15' S, 63° 5'-65° 10' W). Populations of O. puelchana were inferred from sub-fossil deposits (>700 years ago) throughout the gulf, but were documented in surveys a century ago only in the NW coast. The population has declined in the last decades. However, new populations have established recently in the NE and southern regions of the gulf. A Bonamia exitiosa epizootic was coincident with the declining trend of the abundance provided by the time series, suggesting that beds declined as a consequence of parasite infections. Dredging fisheries for scallops took place in the 1970s and 1980s on the NE coast of the gulf, in areas adjacent to the NE oyster beds. We proposed that fishing activities might have had a low impact on oyster beds, since NE beds expanded and increased during that period. The southward expansion of oyster population at latitudes beyond the historical distribution range might reflect long-term adequate environmental conditions for larval survival on the NE and S of the SMG.

Variance in the reproductive success of flat oyster Ostrea edulis L. assessed by parentage analyses in natural and experimental conditions.

In order to document further the phenomena of variance in reproductive success in natural populations of the European flat oyster Ostrea edulis, two complementary studies based on natural and experimental populations were conducted. The first part of this work was focused on paternity analyses using a set of four microsatellite markers for larvae collected from 13 brooding females sampled in Quiberon Bay (Brittany, France). The number of individuals contributing as the male parent to each progeny assay was highly variable, ranging from 2 to more than 40. Moreover, paternal contributions showed a much skewed distribution, with some males contributing to 50-100% of the progeny assay. The second part of this work consisted of the analysis of six successive cohorts experimentally produced from an acclimated broodstock (62 wild oysters sampled in the Quiberon Bay). Allelic richness was significantly higher in the adult population than in the temporal cohorts collected. Genetic differentiation (F(st) estimates) was computed for each pair of samples and all significant values ranged from 0.7 to 11.9%. A limited effective number of breeders (generally below 25) was estimated in the six temporal cohorts. The study gives first indications of the high variance in reproductive success as well as a reduced effective size, not only under experimental conditions but also in the wild. Surprisingly, the pool of the successive cohorts, based on the low number of loci used, appeared to depict a random and representative set of alleles of the progenitor population, indicating that the detection of patterns of temporal genetic differentiation at a local scale most likely depends on the sampling window.

Branchial lesions associated with abundant apoptotic cells in oysters Ostrea edulis of Galicia (NW Spain).

An experiment to evaluate differences in growth, mortality and disease susceptibility among Ostrea edulis stocks was performed. Five families were produced from each of 4 oyster populations (Irish, Greek and 2 Galician). The spat were transferred to a raft in the Ria de Arousa (Galicia, Spain) for grow-out. Monthly samples of each family were histologically processed from 2001 to 2003. One of the pathological conditions discovered by this study was the occurrence of extensive branchial lesions characterized by haemocytic infiltration and loss of branchial architecture. Furthermore, abundant atypical cells occurred among the haemocytes in the lesions in the branchial connective and epithelial tissues, but rarely in the mantle. These cells were contracted in size with nuclei showing chromatin condensation and fragmentation. Some nuclear chromatin aggregated under the nuclear membranes into crescent shapes, whereas others were uniformly dense. Those characteristics suggested that the cells were apoptotic haemocytes, which was confirmed by transmission electron microscopy (TEM) and by a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay using the Apoptag Kit on paraffin sections. A low prevalence of gill lesions was detected in some, but not all, families of every origin peaking in July 2002 and April 2003. No etiologic agent was identified by either histology or TEM; thus, the cause of the abundance of apoptotic cells remains unclear.