Anthony Moreira

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.

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.

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.

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.

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.

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.

Native and exotic oysters in Brazil: Comparative tolerance to hypercapnia

ABSTRACT 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.: 10,000 &mgr;atm), 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 &mgr;atm), 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 (4,000 &mgr;atm) than C. brasiliana. Adult C. gigas showed biochemical alterations mostly in response to high hypercapnia (10,000 &mgr;atm), 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. HIGHLIGHTSC. gigas and C. brasiliana showed differentiated biochemical response to hypercapnia.Juveniles presented a more pronounced stress response than adults in both species.Contrasting metabolic shifts were observed between both species to endure hypercapnia.Antioxidant and metabolic responses were sufficient to prevent excessive LPO.

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 β, 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.