Native and exotic oysters in Brazil: Comparative tolerance to hypercapnia.

Environmental hypercapnia in shallow coastal marine ecosystems can be exacerbated by increasing levels of atmospheric CO2. In these ecosystems organisms are expected to become increasingly subjected to pCO2 levels several times higher than those inhabiting ocean waters (e.g.: 10,000µatm), but still our current understanding on different species capacity to respond to such levels of hypercapnia is limited. Oysters are among the most important foundation species inhabiting these coastal ecosystems, although natural oyster banks are increasingly threatened worldwide. In the present study we studied the effects of hypercapnia on two important oyster species, the pacific oyster C. gigas and the mangrove oyster C. brasiliana, to bring new insights on different species response mechanisms towards three hypercapnic levels (ca. 1,000; 4,000; 10,000 µatm), by study of a set of biomarkers related to metabolic potential (electron transport system - ETS), antioxidant capacity (SOD, CAT, GSH), cellular damage (LPO) and energetic fitness (GLY), in two life stages (juvenile and adult) after 28 days of exposure. Results showed marked differences between each species tolerance capacity to hypercapnia, with contrasting metabolic readjustment strategies (ETS), different antioxidant response capacities (SOD, CAT, GSH), which generally allowed to prevent increased cellular damage (LPO) and energetic impairment (GLY) in both species. Juveniles were more responsive to hypercapnia stress in both congeners, and are likely to be most sensitive to extreme hypercapnia in the environment. Juvenile C. gigas presented more pronounced biochemical alterations at intermediate hypercapnia (4,000µatm) than C. brasiliana. Adult C. gigas showed biochemical alterations mostly in response to high hypercapnia (10,000µatm), while adult C. brasiliana were less responsive to this environmental stressor, despite presenting decreased metabolic potential. Our data bring new insights on the biochemical performance of two important oyster species, and suggest that the duration of extreme hypercapnia events in the ecosystem may pose increased challenges for these organisms as their tolerance capacity may be time limited.

Combined effects of arsenic, salinity and temperature on Crassostrea gigas embryotoxicity.

The combined effects of different salinity and temperature levels on the toxicity of Arsenic (As) were studied on the embryonic development of the oyster Crassostrea gigas. A standardized embryotoxicity test was performed to assess the interactive effects of these stressors, in a full factorial design experiment including a range of salinities (15, 19, 24, 28 and 32), temperatures (16, 20, 24, 28 and 32°C) and As concentrations (100, 300, 600, 1200, 2400µgL-1). The embryotoxicity endpoint was about the determination of normal larvae development rates at various conditions, and median effect concentration (EC50) determination for each As exposure condition. Results showed that toxicity induced by As was characterized by retardation of embryonic development observing toxic effects at lower concentrations than previously reported studies. The presence of As in seawater resulted in a narrower range of tolerance to both salinity and temperature. These findings bring new insights on the impacts of a common contaminant on an important shellfish species having a planktonic early life stage development, with potential implications for population survival and ecosystem functioning in a changing environment.

Comparative sensitivity of Crassostrea angulata and Crassostrea gigas embryo-larval development to As under varying salinity and temperature.

Oysters are a diverse group of marine bivalves that inhabit coastal systems of the world's oceans, providing a variety of ecosystem services, and represent a major socioeconomic resource. However, oyster reefs have become inevitably impacted from habitat destruction, overfishing, pollution and disease outbreaks that have pushed these structures to the break of extinction. In addition, the increased frequency of climate change related events promise to further challenge oyster species survival worldwide. Oysters' early embryonic development is likely the most vulnerable stage to climate change related stressors (e.g. salinity and temperature shifts) as well as to pollutants (e.g. arsenic), and therefore can represent the most important bottleneck that define populations' survival in a changing environment. In light of this, the present study aimed to assess two important oyster species, Crassostrea angulata and Crassostrea gigas embryo-larval development, under combinations of salinity (20, 26 and 33), temperature (20, 24 and 28 °C) and arsenic (As) exposure (0, 30, 60, 120, 240, 480, 960 and 1920 µg. As L-1), to infer on different oyster species capacity to cope with these environmental stressors under the eminent threat of climate change and increase of pollution worldwide. Results showed differences in each species range of salinity and temperature for successful embryonic development. For C angulata, embryo-larval development was successful at a narrower range of both salinity and temperature, compared to C. gigas. Overall, As induced higher toxicity to C. angulata embryos, with calculated EC50 values at least an order of magnitude lower than those calculated for C. gigas. The toxicity of As (measured as median effective concentration, EC50) showed to be influenced by both salinity and temperature in both species. Nonetheless, salinity had a greater influence on embryos' sensitivity to As. This pattern was mostly noticed for C. gigas, with lower salinity inducing higher sensitivity to As. Results were discussed considering the existing literature and suggest that C. angulata populations are likely to become more vulnerable under near future predictions for temperature rise, salinity shifts and pollution.

Impacts of the combined exposure to seawater acidification and arsenic on the proteome of Crassostrea angulata and Crassostrea gigas.

Proteomic analysis was performed to compare the effects of Arsenic (As), seawater acidification (Low pH) and the combination of both stressors (Low pH + As) on Crassostrea angulata and Crassostrea gigas juveniles in the context of global environmental change. This study aimed to elucidate if two closely related Crassostrea species respond similarly to these environmental stressors, considering both single and combined exposures, to infer if the simultaneous exposure to both stressors induced a differentiated response. Identification of the most important differentially expressed proteins between conditions revealed marked differences in the response of each species towards single and combined exposures, evidencing species-related differences towards each experimental condition. Moreover, protein alterations observed in the combined exposure (Low pH + As) were substantially different from those observed in single exposures. Identified proteins and their putative biological functions revealed an array of modes of action in each condition. Among the most important, those involved in cellular structure (Actin, Atlastin, Severin, Gelsolin, Coronin) and extracellular matrix modulation (Ependymin, Tight junction ZO-1, Neprilysin) were strongly regulated, although in different exposure conditions and species. Data also revealed differences regarding metabolic modulation capacity (ATP ß, Enolase, Aconitate hydratase) and oxidative stress response (Aldehyde dehydrogenase, Lactoylglutathione, Retinal dehydrogenase) of each species, which also depended on single or combined exposures, illustrating a different response capacity of both oyster species to the presence of multiple stressors. Interestingly, alterations of piRNA abundance in C. angulata suggested genome reconfiguration in response to multiple stressors, likely an important mode of action related to adaptive evolution mechanisms previously unknown to oyster species, which requires further investigation. The present findings provide a deeper insight into the complexity of C. angulata and C. gigas responses to environmental stress at the proteome level, evidencing different capacities to endure abiotic changes, with relevance regarding the ecophysiological fitness of each species and competitive advantages in a changing environment.

Biochemical alterations in native and exotic oyster species in Brazil in response to increasing temperature.

The increase of temperature in marine coastal ecosystems due to atmospheric greenhouse gas emissions is becoming an increasing threat for biodiversity worldwide, and may affect organisms' biochemical performance, often resulting in biogeographical shifts of species distribution. At the same time, the introduction of non-native species into aquatic systems also threatens biodiversity and ecosystem functions. Oysters are among the most valuable socio economic group of bivalve species in global fishery landings, and also provide numerous ecosystem services. However, the introduction of non-native oyster species, namely Crassostrea gigas for aquaculture purposes may threaten native oyster species, mainly by out competing their native congeners. It is therefore of upmost importance to understand physiological and biochemical responses of native and introduced oyster species in a scenario of global temperature rise, in order to provide knowledge that may allow for better species management. Hence, we compared biochemical alterations of the introduced C. gigas and the native Crassostrea brasiliana, the most important oyster species in Brazil, in response to different thermal regimes for 28days (24, 28 and 32°C). For this, metabolism (ETS), energy content (GLY), antioxidant system (SOD, CAT and GSH/GSSG) and cellular damage (LPO) were assessed in adult and juvenile specimens of both species. Juvenile C. gigas were the most affected by increased temperatures, presenting higher mortality, more pronounced antioxidant response (SOD), whereas adults were more tolerant than juveniles, showing no mortality, no significant changes in antioxidant enzymes activity neither energy expenditure. Native C. brasiliana juveniles presented lower mortality and less pronounced biochemical alterations were noted at higher temperature comparing to non-native C. gigas juveniles. Adult C. brasiliana were the least responsive to tested temperatures. Results obtained in this study bring interesting new insights on different oyster species life stages' physiological and biochemical tolerance towards thermal stress. The native species C. brasiliana showed ability to maintain biochemical performance at higher temperatures, with less pronounced biochemical changes than the non-native species. The introduced (C. gigas) showed to be more sensitive, presenting biochemical alterations to cope with the increase of temperature. Despite the lower observed fitness of the introduced species to temperatures closer to those naturally experienced by the native species, the ability of C. gigas to cope with higher temperatures should still raise concerns towards the native species C. brasiliana management and protection.

Physiological and biochemical impacts induced by mercury pollution and seawater acidification in Hediste diversicolor.

The present study evaluated the impacts of predicted seawater acidification and Hg pollution, when stressors were acting alone and in combination, on the polychaete Hediste diversicolor. Polychaetes were exposed during 28days to low pH (7.5), Hg (5µg/L) and pH7.5+Hg, and physiological alterations (respiration rate), biochemical markers related to metabolic potential (glycogen and protein content, electron transport system activity) and oxidative status (activity of antioxidant and biotransformation enzymes, lipid peroxidation) were evaluated. The results obtained clearly showed that polychaetes were sensitive to low pH and Hg contamination, both acting alone or in combination. Organisms used their energy reserves under stressful conditions, which decreased by up to half of the control content, probably to fuel defence mechanisms. Our findings further demonstrated that polychaetes exposed to these stressors presented increased antioxidant defence mechanisms (3 fold compared to control). However, organisms were not able to prevent cellular damage, especially noticed at Hg exposure and pH7.5. Overall, although all the tested conditions induced oxidative stress in Hediste diversicolor, the combined effect of seawater acidification and Hg contamination did not induce higher impacts in polychaetes than single stressor exposures. These findings may indicate that predicted climate change scenarios may not increase Hediste diversicolor sensitivity towards Hg and may not significantly change the toxicity of this contaminant to this polychaete species.

The effects of arsenic and seawater acidification on antioxidant and biomineralization responses in two closely related Crassostrea species.

Ocean acidification processes are major threats to marine calcifying organisms, mostly affecting biomineralization related processes. Abiotic stressors acting on marine systems do not act alone, rather in a combination of multiple stressors, especially in coastal habitats such as estuaries, where anthropogenic and environmental pressures are high. Arsenic (As) is a widely distributed contaminant worldwide and its toxicity has been studied on a variety of organisms. However, the effect of low pH on the toxicity of As on marine organisms is unknown. Here, we studied the combined effects of ocean acidification and As exposure on two closely related oyster species (Crassostrea angulata and Crassostrea gigas), by use of a biochemical approach. Oxidative stress related parameters were studied along with the assessment of biomineralization enzymes activity after 28days of exposure. Results showed that both species were sensitive to all tested conditions (low pH, As and pH+As), showing enhancement of antioxidant and biotransformation defenses and impairment of biomineralization processes. Glutathione S-transferases (GSTs) activity were significantly higher in oysters exposed to As, showing activation of detoxification mechanisms, and a lower GSTs activity was observed in low pH+As condition, indicating an impact on the oysters capacity to detoxify As in a low pH scenario. Carbonic anhydrase (CA) activity was significantly lower in all tested conditions, showing to be affected by both As and low pH, whereas the combined effect of low pH+As was not different from the effect of low pH alone. Multivariate analysis of biochemical data allowed for the comparison of both species performance, showing a clear distinction of response in both species. C. gigas presented overall higher enzymatic activity (GSTs; superoxide dismutase; catalase; CA and acid phosphatase) and higher cytosolic GSH content in As exposed oysters than C. angulata. Results obtained indicate a higher tolerance capacity of the Pacific oyster C. gigas towards the tested conditions.

Salinity influences the biochemical response of Crassostrea angulata to Arsenic.

The increasing rate of occurrence and persistence of climatic events causing salinity shifts, in combination with contamination, may further challenge organisms response to environmental stress. Hence, we studied the effects of different salinity levels (10, 20, 30 and 40) on the response of the oyster Crassostrea angulata to Arsenic (As) exposure (4 mg L(-1)). Total As, Na(+) and K(+) concentrations in oyster tissues were determined. Biochemical analysis were performed to assess osmotic regulation (CA), metabolism (ETS), enzymatic (SOD, CAT and GSTs) and non-enzymatic (GSH/GSSG and LPO) markers of oxidative stress. Results obtained showed significantly higher metabolic activities in oysters maintained in low salinity (10) exposure, coupled with higher As accumulation, as well as higher SOD and CAT activities, compared to higher salinities (30 and 40). GSTs activity and LPO levels were higher in oysters exposed to As at salinities 20, 30 and 40, compared to the same conditions without As. From our findings we concluded that the response of C. angulata to As is influenced by salinity. At the lowest salinity (10) oysters accumulated higher As concentrations, here attributed to higher metabolic rate involved in physiological osmoregulation, also stimulating antioxidant related enzymes activity (SOD and CAT) and thus preventing increased LPO (higher ETS activity also observed without As). On the contrary, at salinities 30 and 40 with As, antioxidant SOD and CAT were inhibited, enabling for LPO generation. Given our results, the effects of As on the oysters antioxidant capacity appears to be more deleterious under higher salinities (20, 30 and 40), comparing to salinity 10. The differentiated responses demonstrated in the present study in C. angulata oysters exposed to As under different salinities, bring new insights on the mechanisms of environmental adaptability of this species, namely to salinity shifts, and the interactions between such alterations and As exposure.

Biochemical alterations induced in Hediste diversicolor under seawater acidification conditions.

Seawater pH is among the environmental factors controlling the performance of marine organisms, especially in calcifying marine invertebrates. However, changes in non-calcifying organisms (including polychaetes) may also occur due to pH decrease. Polychaetes are often the most abundant group of organisms in estuarine systems, representing an important ecological and economic resource. Thus, the present study aimed to evaluate the impacts of seawater acidification in the polychaete Hediste diversicolor, a species commonly used as bioindicator. For this, organisms were exposed to different pH levels (7.9, 7.6 and 7.3) during 28 days and several biochemical markers were measured. The results obtained demonstrated that pH decrease negatively affected osmotic regulation and polychaetes metabolism, with individuals under low pH (7.6 and 7.3) presenting higher carbonic anhydrase activity, lower energy reserves (protein and glycogen content) and higher metabolic rate (measured as Electron transport system activity). The increase on CA activity was associated to organisms osmoregulation capacity while the increase on ETS and decrease on energy reserves was associated to the polychaetes capacity to develop defense mechanisms (e.g. antioxidant defenses). In fact, despite having observed higher lipid peroxidation at pH 7.6, in polychaetes at the lowest tested pH (7.3) LPO levels were similar to values recorded in individuals under control pH (7.9). Such findings may result from higher antioxidant enzyme activity at the lowest tested pH, which prevented organisms from higher oxidative stress levels. Overall, our study demonstrated how polychaetes may respond to near-future ocean acidification conditions, exhibiting the capacity to develop biochemical strategies which will prevent organisms from lethal injuries. Such defense strategies will contribute for polychaetes populations maintenance and survival under predicted seawater acidification scenarios.

The impacts of pharmaceutical drugs under ocean acidification: New data on single and combined long-term effects of carbamazepine on Scrobicularia plana.

Ocean acidification and increasing discharges of pharmaceutical contaminants into aquatic systems are among key and/or emerging drivers of environmental change affecting marine ecosystems. A growing body of evidence demonstrates that ocean acidification can have direct and indirect impacts on marine organisms although combined effects with other stressors, namely with pharmaceuticals, have received very little attention to date. The present study aimed to evaluate the impacts of the pharmaceutical drug Carbamazepine and pH 7.1, acting alone and in combination, on the clam Scrobicularia plana. For this, a long-term exposure (28 days)was conducted and a set of oxidative stress markers was investigated. The results obtained showed that S. plana was able to develop mechanisms to prevent oxidative damage when under low pH for a long period, presenting higher survival when exposed to this stressor compared to CBZ or the combination of CBZ with pH 7.1. Furthermore, the toxicity of CBZ on S. plana was synergistically increased under ocean acidification conditions (CBZ + pH 7.1): specimens survival was reduced and oxidative stress was enhanced when compared to single exposures. These findings add to the growing body of evidence that ocean acidification will act to increase the toxicity of CBZ to marine organisms,which has clear implications for coastal benthic ecosystems suffering chronic pollution from pharmaceutical drugs.

The effects of water acidification, temperature and salinity on the regenerative capacity of the polychaete Diopatra neapolitana.

Changes in seawater pH, temperature and salinity are expected to occur in the near future, which can be a threat to aquatic systems, mainly for marine coastal areas, and their inhabiting species. Hence, the present study proposes to evaluate the effects of temperature shifts, pH decrease and salinity changes in the tissue's regenerative capacity of the polychaete Diopatra neapolitana. This study evidenced that D. neapolitana individuals exposed to lower pH exhibited a significantly lower capacity to regenerate their body, while with the increase of temperature individuals showed a higher capacity to regenerate their tissues. Furthermore, the present work demonstrated that individuals exposed to salinities 28 and 35 did not present significant differences between them, while salinities 21 and 42 negatively influenced the regenerative capacity of D. neapolitana. At the end of regeneration, comparing all conditions, high salinity (42) seemed to have a greater impact on the regenerative capacity of individuals than the other factors, since under this condition individuals took longer to completely regenerate. Overall, this study demonstrated that variations in abiotic factors can strongly affect D. neapolitana's performance.

How life history influences the responses of the clam Scrobicularia plana to the combined impacts of carbamazepine and pH decrease.

In the present study, the bivalve Scrobicularia plana, collected from two contrasting areas (pristine location and mercury contaminated area), was selected to assess the biochemical alterations imposed by pH decrease, carbamazepine (an antiepileptic) and the combined effect of both stressors. The effects on oxidative stress related biomarkers after 96 h exposure revealed that pH decrease and carbamazepine induced alterations on clams, with greater impacts on individuals from the contaminated area which presented higher mortality, higher lipid peroxidation and higher glutathione S-transferase activity. These results emphasize the risk of extrapolating results from one area to another, since the same species inhabiting different areas may be affected differently when exposed to the same stressors. Furthermore, the results obtained showed that, when combined, the impact of pH decrease and carbamazepine was lower than each stressor acting alone, which could be related to the defence mechanism of valves closure when bivalves are under higher stressful conditions.

Venerupis decussata under environmentally relevant lead concentrations: Bioconcentration, tolerance, and biochemical alterations.

The edible clam Venerupis decussata is widely distributed in European aquatic systems, some of which are under strong anthropogenic pressure, which can contribute to trophic transfer of xenobiotics to humans. Accordingly, the present study focused on the tolerance, bioconcentration, and biochemical responses of V. decussata after exposure to ecologically relevant concentrations of lead. Health risks to humans after consumption of clams was also explored. An acute toxicity assay (96 h) was conducted with wild clams, using Pb exposure concentrations ranging from 0 mg L(-1) to 1.80 mg L(-1). Lethality, bioconcentration factor (BCF), intracellular partitioning, and a relevant set of biomarkers were used as endpoints. Clams, interstitial water, water column, and sediment samples were collected to analyze Pb concentration. The Pb concentration in wild clams was below international consumption guidelines. Under laboratory conditions, clams revealed high sensitivity to Pb (median lethal concentration of 0.65 mg L(-1)), with a high bioconcentration ability (bioconcentration factor > 1) during exposure. The intracellular partitioning data showed that most of the Pb had accumulated in the insoluble fraction (>80%). Several significant biochemical changes were observed, namely on catalase and glutathione-S-tranferase activities and metalothionein content. Overall, it was demonstrated that the European clam has a reduced tolerance to Pb, compared with other bivalves. However, consumption of clams from the Ria de Aveiro lagoon (Portugal) does not raise public health concerns in terms of Pb.