Oxidative effects of inorganic and organic contaminants on haemolymph of mussels

We applied a newly-established method in haemolymph of mussels, Mytilus galloprovincialis, exposed to different concentrations of heavy metals, such as zinc and cadmium and organic pollutants, such as PAHs and lindane, for the detection of total antioxidant capacity (TAC). The susceptibility of exposed mussels was increased in relation to oxidative stress induced by contaminants tested. Oxidative modifications of proteins were estimated by measuring protein carbonyl content (PCC) and malondialdehyde levels (MDA). For PCC measurement, a highly sensitive and accurate ELISA method, which requires only 5 µg of protein, was used. The significant increase of PCC and MDA in haemolymph of exposed mussels reinforces its role as biomarkers of oxidative stress. Significant correlation of TAC assay, PCC and MDA was conducted in order to evaluate the utility of PCC and TAC assay, used in the present study, as tools for determining oxidative effects of pollutants in mussels. The results reinforce the application of PCC method as useful tool for the determination of PCC alterations in haemolymph of mussels exposed to different levels of contaminants. In addition, the TAC method gives encouraging results, concerning its ability to predict antioxidant efficiency in haemolymph of mussels exposed to inorganic and organic contaminants.     

Physiological and proteomic responses in Mytilus edulis exposed to PCBs and PAHs extracted from Baltic Sea sediments

Stress responses in blue mussels (Mytilus edulis. L.) exposed to rganic pollutants were measured using several physiological measures and as changes in protein expression. Blue mussels from the Baltic Sea were exposed for 6 days in a flow-through system to two fractions of extracted Baltic sediments (containing primarily PAHs or PCBs) from one industrially impacted site and one off-shore site. Exposure to Aroclor1248 (a commercial PCB mixture) was included as a reference treatment. Physiological response was measured as changes in respiration, excretion, clearance rates and scope for growth. Of the physiological responses, only clearance rate and scope for growth in the Aroclor and impacted site PCB treatments differed significantly (p<0.05) from control organisms, perhaps due to a large variation among individuals. Seven proteins were observed, presumed to be from stress protein families (hsp60, hsp70 and hsp90) on one-dimensional electrophoresis gels. All protein levels, except three proteins, 62, 73 and 90 kDa, in response to PCB exposure from the industrial site, were significantly higher (p<0.05) in treated than in control organisms, suggesting the use of stress-inducible proteins as diagnostics in risk assessment. A wider sample of proteins was observed using two-dimensional gel electrophoresis. The presence or absence of protein spots compared to control organisms was used as an indication of stress. Between 23 and 76 proteins or spots were present and 15 to 23 absent compared to controls, and the results supported the physiological and one-dimensional gel results, suggesting that the mussels were indeed suffering from stress. The methods used here represent stress monitoring at two different levels of biological organization; the cellular- and the level of individual organisms. In this experiment the protein response showed less variation among individuals compared to the physiological parameters. The protein response, however, still suffers from the lack of interpretation into commonly used monitoring terms, which emphasizes the need for more knowledge of whether the response is a momentary reflection of exposure or an early warning of higher order effects.     

Lipid and fatty acid biomarkers as proxies for environmental contamination in caged mussels Mytilus galloprovincialis

Mussels (Mytilus galloprovincialis) were transplanted from a reference site (Syracuse harbour) to an impacted site (Augusta Bay) from January to July 2013 to assess the biochemical response of caged mussels to high trace element and polycyclic aromatic hydrocarbon (PAH) contamination, using lipid and fatty acid (FA) biomarkers. Sediment and mussels were analysed to assess contaminant bioavailability in the study sites and bioaccumulation in mussel tissue. Trace elements (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, V, Zn) and PAHs were significantly higher in mussels from Augusta than in those from Syracuse, mirroring the different environmental contamination. The biological impact quotient (BIQs), which indicates the potential biological impact of mussel-accumulated contaminants, reflected the highest contaminant concentrations found in Augusta and their temporal trend, which increased from the start of the experiment to 3–4 months after transplanting, followed by a decrease, as indication of mussel detoxification processes. Lower condition index (CI) and phospholipids (PL), as well as higher total (TL) and neutral lipids (NL) in mussels from Augusta, indicated the occurrence of a physiological and biochemical stress response to pollutant exposure and accumulation. Differences in FA composition, especially polyunsaturated (PUFA), essential (EFA) and non-methylene interrupted dienoic (NMID) FAs between the study sites revealed the occurrence of stress-induced lipid peroxidation, followed by compensatory/adaptive processes in Augusta mussels. The marked increase in bacterial biomarkers, mainly cyclopropyl (CY) FAs, reflecting the greater bioaccumulation of chemicals in the Augusta mussels, may be the result of bacterial infections or symbiosis with bacteria involved in detoxification processes.     

Seasonal effects of sun exposure and emersion on intertidal seaweed physiology: Fluctuations in antioxidant contents, photosynthetic pigments and photosynthetic efficiency in the red alga Porphyra umbilicalis Kützing (Rhodophyta, Bangiales)

There is a great deal of speculation regarding the physiological and biochemical mechanisms that give certain seaweed species the ability to colonize the intertidal zone. Frequent exposure to ambient temperatures and high irradiance levels in addition to dehydration during tidal emersion generates acute physiological stress. The ability of seaweeds like Porphya to overcome these challenges and survive in such a harsh environment has been linked to elevated reactive oxygen metabolism. The current study focused on measuring seasonal changes in antioxidant enzymes plus alterations in pigment contents and photosynthetic efficiency of P. umbilicalis plants found growing in the uppermost intertidal zone.Our results suggest that P. umbilicalis exhibits increased antioxidant metabolism, which could contribute to its success in colonizing such a stressful habitat. Elevated levels of glutathione reductase GTR, catalase and carotenoid contents during emersion suggested heightened protection against reactive oxygen species ROS damage is a necessary attribute for species in the upper intertidal regions. This hypothesis was further strengthened by the finding that the greatest antioxidant increases were observed during summer months when irradiance levels and temperatures were at their peak. Winter emersion did not elicit the same physiological response, as antioxidant levels were similar in submersed and emersed plants.For the most part, photosynthetic pigments were largely affected by sun exposure and less by emersion stress. Shaded blades maintained higher concentrations of photosynthetic pigments compared to sun exposed thalli concurring with established research. Photosynthetic efficiency measurements indicated emersion and not sun exposure was the greater facilitator of photoinhibitory damage and ROS generation at PSII. The findings of this field study strengthen previous assertions that protection via elevated antioxidant metabolism and increased PSII repair are involved in providing relief from the acute environmental stresses in the intertidal zone.     

Physiological responses to emersion in the intertidal green crab,Carcinus maenas (L.)

The green shore crab, Carcinus maenas, undergoes on average 6?h periods of emersion during each low-tide cycle during the summer months. Under those conditions, the crab is cut off from its normal water environment and is exposed to potential stress from a suite of environmental and physiological changes: dehydration, compromised gas exchange and resultant internal hypoxia and hypercapnia, thermal stress, and ammonia toxicity. This study examined the comprehensive responses of the green crab in water and to a 6?h emersion period laboratory simulation of a tidal cycle followed by a 1?h re-immersion period, measuring indicators of dehydration, hemolymph osmolality, oxygen uptake, hemolymph acid–base status, heart and ventilatory rate, and hemolymph ammonia and ammonia excretion. Green crabs showed physiological responses of varying magnitude to 6?h of emersion. Individuals were found in the field exclusively under rocks and large clumps of seaweed where temperatures were approximately half those of exposed surfaces and relative humidity was about twice as high as ambient air. During emersion, crabs lost less than 5% of their wet weight, and hemolymph osmolality did not increase significantly. Oxygen uptake continued in air at about 50% of the control, aquatic values; and the gills continued to be ventilated by the scaphognathite, albeit at lower rates. Hemolymph lactate concentrations increased, indicating a shift to a greater reliance on anaerobic metabolism to support energetic needs. A slight acidosis developed in the hemolymph after 1?h of emersion, but it did not increase thereafter. Ammonia concentrations in the hemolymph were unchanged, as ammonia was volatilized by the gills and excreted into the air as NH₃ gas. These results show that the green crab copes with emersion by seeking refuge in microhabitats that mitigate the changes in the physical parameters of intertidal emersion. Physiologically, desiccation is avoided, cardio-respiratory processes are maintained at reduced levels, and hemolymph acid–base balance is minimally affected. Ammonia toxicity appears to be avoided by a shift to excreting NH₃ gas directly or indirectly to air.     

Lack of physiological responses to hydrocarbon accumulation by Mytilus trossulus after 3–4 years chronic exposure to spilled Exxon Valdez crude oil in Prince William Sound

Mussels, Mytilus trossulus, were sampled in 1992 and 1993 from beaches in Prince William Sound that had been oiled by the Exxon Valdez spill of March, 1989. At some of the oiled beaches, mussels were collected from beds overlying oiled sediments, and from bedrock adjacent to these beds. Mussels were also collected from beaches within the Sound that had not been impacted by the spill. Polynuclear aromatic hydrocarbon (PAH) concentrations in mussel tissue, physiological responses (byssal thread production, condition index, clearance rate, and glycogen content), were determined for each group of mussels. Total PAH concentrations in mussel tissue ranged from 0 to 6 μg g⁻¹, and were significantly greater in mussels from oiled beds than those from reference beds. No significant differences were noted in byssal thread production, condition index, clearance rate, or glycogen content between oiled sample sites and reference sites. The lack of physiological response was surprising because mussels in this study were chronically exposed to PAH for 3–4 years, and none of the physiological responses measured appeared to be affected by that exposure. The lack of a physiological response suggests that chronically exposed mussels may develop a physiological tolerance to PAH, but we recognize that these measures may not have been sensitive enough to discriminate response from background noise.     

Linking Physiology To Ecological Function: Environmental Conditions Affect Performance And Size Of The Intertidal Kelp Hedophyllum Sessile (Laminariales,Phaeophyceae)

For autogenic ecosystem engineers, body size is an aspect of individual performance that has direct connections to community structure; yet the complex morphology of these species can make it difficult to draw clear connections between the environment and performance. We combined laboratory experiments and field surveys to test the hypothesis that individual body size was determined by disparate localized physiological responses to environmental conditions across the complex thallus of the intertidal kelp Hedophyllum sessile, a canopy‐forming physical ecosystem engineer. We documented substantial (> 40%) declines in whole‐thallus photosynthetic potential (as Maximum Quantum Yield, MQY) as a consequence of emersion, which were related to greater than 10‐fold increases in intra‐thallus MQY variability (as Coefficient of Variation). In laboratory experiments, desiccation and high light levels during emersion led to lasting impairment of photosynthetic potential and an immediate > 25% reduction in area due to tissue contraction, which was followed by complete loss of structural integrity after three days of submersion. Tissue exposed to desiccation and high light during emersion had higher nitrogen concentrations and lower phlorotannin concentrations than tissue in control treatments (on average 1.36 and 0.1x controls, respectively), suggesting that conditions during emersion have the potential to affect food quality for consumers. Our data indicate that the complex thallus morphology of H. sessile may be critical to this kelp’s ability to persist in the intertidal zone despite the physiological challenges of emersion and encourage a more nuanced view of the concept of “sub‐lethal stress” on the scale of the whole individual.     

Direct effects of physical stress can be counteracted by indirect benefits: oyster growth on a tidal elevation gradient

The paradigmatic gradient for intertidal marine organisms of increasing physical stress from low to high elevation has long served as the basis for using direct effects of duration of water coverage to predict many biological patterns. Accordingly, changes in potential feeding time may predict the direction and magnitude of differences between elevations in individual growth rates of sessile marine invertebrates. Oysters (triploid Crassostrea ariakensis) experimentally introduced at intertidal (MLW+0.05 m) and subtidal (MLW–0.25 m) elevations in racks provided a test of the ability to use duration of water coverage to predict changes in growth. During early-to-mid winter, a depression of 38–47% in shell growth of intertidal oysters matched the 36% reduction in available feeding time relative to subtidal oysters. In late winter as solar heating of exposed oysters increased, growth differences of 52–55% departed only slightly from the predicted 39%. In spring, however, duration of water coverage failed to predict even the correct direction of growth change with elevation as intertidal oysters grew 34% faster despite 39% less feeding time. Intense seasonal development of shell fouling by other suspension feeders like ascidians, mussels, and barnacles on subtidal (94% incidence) but not on aerially exposed intertidal (21–38% incidence) oysters may explain why duration of water cover failed to predict spring growth differences. Less intense fouling develops on intertidal oysters due to the physiological stress of aerial exposure on settlers, especially during higher temperatures and longer solar exposures of spring. Fouling by suspension feeders is known to reduce growth of the host through localized competition for food and added energetic costs. Thus, in springtime, indirect effects of aerial exposure providing a partial refuge from biological enemies overwhelmed direct effects of reduced duration of water coverage to reverse the expected pattern of slower intertidal growth of a marine invertebrate.     

Emersion Induces Nitrogen Release and Alteration of Nitrogen Metabolism in the Intertidal Genus Porphyra

We investigated emersion-induced nitrogen (N) release from Porphyra umbilicalis Kütz. Thallus N concentration decreased during 4 h of emersion. Tissue N and soluble protein contents of P. umbilicalis were positively correlated and decreased during emersion. Growth of P. umbilicalis did not simply dilute the pre-emersion tissue N concentration. Rather, N was lost from tissues during emersion. We hypothesize that emersion-induced N release occurs when proteins are catabolized. While the δ¹⁵N value of tissues exposed to emersion was higher than that of continuously submerged tissues, further discrimination of stable N isotopes did not occur during the 4 h emersion. We conclude that N release from Porphyra during emersion did not result from bacterial denitrification, but possibly as a consequence of photorespiration. The release of N by P. umbilicalis into the environment during emersion suggests a novel role of intertidal seaweeds in the global N cycle. Emersion also altered the physiological function (nitrate uptake, nitrate reductase and glutamine synthetase activity, growth rate) of P. umbilicalis and the co-occurring upper intertidal species P. linearis Grev., though in a seasonally influenced manner. Individuals of the year round perennial species P. umbilicalis were more tolerant of emersion than ephemeral, cold temperate P. linearis in early winter. However, the mid-winter populations of both P. linearis and P. umbilicalis, had similar temporal physiological patterns during emersion.     

Seasonal variations of a battery of biomarkers and physiological indices for the mussel Mytilus galloprovincialis transplanted into the northwest Mediterranean Sea

Seasonal variations of six mussel (Mytilus galloprovincialis) biomarkers at two sites in the Mediterranean Sea were compared with physiological indices (condition, growth and gonad maturation), environmental parameters (temperature, salinity and turbidity), and chemical contamination levels. The basal levels of acetylcholinesterase (AChE), DNA adducts, benzo[a]pyrene hydroxylase (BPH), heat-shock proteins (HSP70), metallothioneins (MT) and P-glycoprotein (P-gp)-mediated multixenobiotic resistance (MXR) were estimated as early warning signals in caged mussels sampled at Carteau (native site) and La Fourcade (transplantation site) over a 2-year period. The Carteau and La Fourcade mussels have specific chemical contamination profiles but a similar range of values. For example, both are highly contaminated by heavy metals (201 and 258.4 mg kg(-1) dw, respectively) and considered as moderately impacted for polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). However, contamination levels at Carteau are twice as high for PAHs (101.5 mg g(-1) dw) and PCBs (90.2 mg g(-1) dw) than La Fourcade. The seasonal contamination trend at Carteau showed six-fold higher levels of pyrolytic pollutants in winter. Although few tissue lesions were detected in individuals studied at either site, greater parasitic infestation was observed at Carteau. The results of findings from the two Mediterranean pilot studies support the adaptability of transplanted mussels to be used as biomarkers and to establish physiological endpoints for chemical contaminant exposure.     

Environmental contaminants and the prevalence of hemic neoplasia (leukemia) in the common mussel (Mytilus edulis complex) from Puget Sound, Washington, U.S.A

The relationship between hemic neoplasia, a blood cell disorder in bivalve molluscs, and chemical contaminants was evaluated in the common mussel (Mytilus edulis complex). Hemic neoplasia (HN) is endemic to mussel populations in Puget Sound. The prevalence of hemic neoplasia ranged from 0 to 30% in mussels from nine sites in Puget Sound, Washington. Organic chemical contamination in sediment from these sites range from 0.1 to 64.0 ppm of polycyclic aromatic hydrocarbons (PAHs) and 0.07 to 0.50 ppm chlorinated hydrocarbons. No relationship between the body burden of environmental contaminants and the prevalence of HN in mussels was identified. To evaluate the short-term ability of chemical contaminants to induce HN in mussels, mussels, from a site where mussels were previously determined to be HN free, were fed microencapsulated PAHs (composed of a mixture of phenanthrene, flouranthene, and benzo[a]pyrene) or PCBs (Aroclor 1254) and the prevalence of HN was assessed after 30 days of exposure. Although an apparent increase in HN prevalence (20 to 30%) was observed in all treatments groups except the untreated controls, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. A long-term (180-day) exposure study was conducted to evaluate the influence of PAHs or PCBs in modulating the prevalence of HN in a mussel population already exhibiting a moderate HN prevalence. Mussels, from a site where mussels were previously determined to exhibit a background prevalence of HN, fed microencapsulated PAHs, PCBs, and corn oil (vehicle) over a long time period (180 days), revealed an apparent increased prevalence of HN (30 to 40%) above the low levels (20%) initially present. However, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. Although chemical contaminants have been proposed as a modulating factor in the development and promotion of HN in bivalve molluscs from environmentally stressed and degraded habitats, we find no evidence that chemical contaminants induce or promote the development of HN in the mussel M. edulis complex.     

Exposure to solar radiation drives organismal vulnerability to climate: Evidence from an intertidal limpet

Understanding the physiological abilities of organisms to cope with heat stress is critical for predictions of species’ distributions in response to climate change. We investigated physiological responses (respiration and heart beat rate) of the ectotherm limpet Patella vulgata to heat stress events during emersion and the role of seasonal and microclimatic acclimatization for individual thermal tolerance limits. Individuals were collected from 5 microhabitats characterized by different exposure to solar radiation in the high intertidal zone of a semi-exposed rocky shore in winter and summer of 2014. Upper thermal tolerance limits (heat coma temperatures – HCTs, and heart rate Arrhenius break temperatures - ABTs) were determined for individuals from each microhabitat in both seasons under laboratory conditions. While we found a clear seasonal acclimatization, i.e., higher HCTs and ABTs in summer than in winter, we did not find evidence for microhabitat-specific responses that would suggest microclimatic acclimatization. However, operative limpet temperatures derived from in-situ temperature measurements suggest that individuals from sun exposed microhabitats have a much narrower thermal safety margins than those from less exposed surfaces or within crevices. Microhabitat specific thermal safety margins caused by high thermal heterogeneity at small spatial scales and the lack of short term acclimatization will likely shape small scale distribution patterns of intertidal species in response to the predicted increase in the frequency and intensity of heat waves.     

Heat-shock response of the upper intertidal barnacle Balanus glandula: thermal stress and acclimation

In the intertidal zone in the Pacific Northwest, body temperatures of sessile marine organisms can reach 35 degrees C for an extended time during low tide, resulting in potential physiological stress. We used immunochemical assays to examine the effects of thermal stress on endogenous Hsp70 levels in the intertidal barnacle Balanus glandula. After thermal stress, endogenous Hsp70 levels did not increase above control levels in B. glandula exposed to 20 and 28 degrees C. In a separate experiment, endogenous Hsp70 levels were higher than control levels when B. glandula was exposed to 34 degrees C for 8.5 h. Although an induced heat-shock response was observed, levels of conjugated ubiquitin failed to indicate irreversible protein damage at temperatures up to 34 degrees C. With metabolic labeling, we examined temperature acclimation and thermally induced heat-shock proteins in B. glandula. An induced heat-shock response of proteins in the 70-kDa region (Hsp70) occurred in B. glandula above 23 degrees C. This heat-shock response was similar in molting and non-molting barnacles. Acclimation of B. glandula to relatively higher temperatures resulted in higher levels of protein synthesis in the 70-kDa region and lack of an upward shift in the induction temperature for heat-shock proteins. Our results suggest that B. glandula may be well adapted to life in the high intertidal zone but may lack the plasticity to acclimate to higher temperatures.     

Spilled Oils: Static Mixtures or Dynamic Weathering and Bioavailability?

Polynuclear aromatic hydrocarbons (PAHs) from sequestered MV Selendang Ayu oil were biologically available in 2008, 3.6 y after it was spilled along Unalaska Island, Alaska. Thermodynamically driven weathering was the most probable mechanism of organism exposure to PAHs. Alkane and PAH composition in oil changed over time as smaller constituents were preferentially lost, indicative of weathering. In contrast, composition of the largest compounds (biomarkers) including triterpanes, hopanes, and steranes remained unchanged. Smaller molecules (the PAHs) lost from stranded oil were observed in indigenous mussels and passive samplers deployed in July 2008. Concentration and composition of PAHs were significantly different than in a non-oiled reference area and patterns observed in mussels were repeated in passive samplers deployed in three zones (intertidal, subtidal, and water). Thus, hydrocarbons lost from one compartment (sequestered whole oil) were detectable in another (mussels and passive samplers) implying aqueous transfer. Quantities of mobile oil constituents were small, yielding uptake concentrations that are likely inconsequential for mussels, but the sensitivity provided by bioaccumulation and passive sampler uptake ensured that dissolved hydrocarbons were detectable.     

Thermal stress,thermal safety margins and acclimation capacity in tropical shallow waters—An experimental approach testing multiple end-points in two common fish

Tropical organisms are predicted to be among the most impacted by increasing sea surface temperatures, particularly those from intertidal habitats. In this study, a complete thermal biology assessment was conducted for two widespread tropical Atlantic shallow reef fish: Abudefduf saxatilis (damselfish) and Scartella cristata (blenny), which make extensive use of tide pools. The main objectives were to measure the time-course changes during one month in i) thermal and oxidative stress biomarkers (in gills, muscle and skin), ii) upper thermal limits, acclimation capacity and thermal safety margins and iii) body size, condition and energy reserves (total protein and lipid contents), under two temperature treatments (control – mean summer temperature, and elevated temperature − + 3 °C, as projected by climate warming scenarios for the end of this century).Results from biomarker analyses suggest that under increased temperature, both species displayed a typical response of physiological stress characterized by the activation of molecular chaperones and antioxidant protection. Both species presented a significant acclimation potential in the long term, as shown by increased critical thermal maxima values at higher temperature. However, these species may already be at risk during summer heat waves, as thermal safety margins for both species were low. Additionally, despite acclimation, some energetic tradeoffs may exist, since specimens from both species showed smaller body sizes at higher temperature (even though maintaining body condition). Finally, temperature treatments had a significant influence not only in the total amount of energy reserves (lipid contents) but also in their rate of deposition or depletion (total proteins and lipid contents). This is the first multi-end-point holistic approach to assess the impact of warming in shallow tropical water fish and it highlights the high risk that intertidal organisms are facing in both present and future sea surface temperature conditions.     

PHYSIOLOGICAL STRESS AND COLOR POLYMORPHISM IN THE INTERTIDAL SNAIL NUCELLA LAPILLUS

The intertidal snail Nucella lapillus exhibits considerable variation in shell color both within and between populations differentially exposed to wave action. Populations from high-wave-energy shores tended to be highly polymorphic and were dominated by pigmented morphs (especially brown), while those at more sheltered locations exhibited less polymorphism and were predominantly white. Field and laboratory experiments were conducted to determine the role of physiological stress and selective predation in maintaining the observed distribution of color morphs. The results demonstrated that 1) physiological stress from high temperature and desiccation during periods of tidal emersion was greater on protected shores, 2) under similar natural conditions, brown morphs heated up faster, attained higher temperatures, desiccated more rapidly, and suffered greater mortality than did white morphs, and 3) when pairs of brown and white morphs were tethered intertidally there was virtually no mortality of either morph on the exposed shore or in shaded microhabitats on the protected shore, but brown morphs suffered much greater mortality in sunny microhabitats on the protected shore. These findings demonstrate that the interpopulation variation in shell color of N. lapillus is in part a response to a selective gradient in physiological stress. Selection for crypsis by visually hunting predators did not appear to play a prominent role; however, only adults were considered, and the predation experiments were conducted in the fall before shorebirds that prey on whelks had arrived from their summer feeding grounds. Further experimentation to quantify the effects of visual predators such as birds and fish, particularly on juvenile snails, is necessary to assess adequately the importance of predation.     

Effects of seawater acidification and salinity alterations on metabolic,osmoregulation and oxidative stress markers in Mytilus galloprovincialis

The impacts of seawater acidification and salinity shifts on metabolism, energy reserves, and oxidative status of mussels have been largely neglected. With the aim to increase the current knowledge for the mussel Mytilus galloprovincialis a 28-day chronic test was conducted during which mussels were exposed to two pH (7.8 and 7.3; both at control salinity 28) and three salinity (14, 28 and 35, at control pH, 7.8) levels. After exposure to different conditions, mussels electron transport system activity, energy reserves (protein and glycogen content) carbonic anhydrase activity, antioxidant defences and cellular damage were measured. Results obtained showed that mussels exposed to seawater acidification presented decreased metabolic capacity that may have induced lower energy expenditure (observed in higher glycogen, protein and lipids content at this condition). Low pH condition induced the increase of carbonic anhydrase activity that was related to acid-base balance, while no significant activation of antioxidant defence mechanisms was observed resulting in higher LPO. Regarding the impacts of salinity, the present study showed that at the highest salinity (35) mussels presented lower metabolic activity (also related to lower energetic expenditure) and an opposite response was observed at salinity 14. Carbonic anhydrase slightly increased at stressful salinity conditions, a mechanism of homeostasis maintenance. Lower metabolic activity at the highest salinity, probably related to valves closure, helped to mitigate the increase of LPO in this condition. At low salinity (14), despite an increase of antioxidant enzymes activity, LPO increased, probably as a result of ROS overproduction from higher electron transport system activity. The present findings demonstrated that Mytilus galloprovincialis oxidative status and metabolic capacity were negatively affected by low pH and salinity changes, with alterations that may lead to physiological impairments namely on mussels reproductive output, growth performance and resistance to disease, with ecological and economic implications.Indicators: Physiological and biochemical changes in Mytilus galloprovincialis in response to low pH and salinity changes     

Differential pattern of Cu/Zn superoxide dismutase isoforms in relation to tidal spatio-temporal changes in the blue mussel Mytilus edulis

Inducible antioxidant defences in marine organisms such as mussel bivalves are commonly used as biomarkers of pollutant-induced oxidative stress and their variations proposed as one of the biological effect measurements for assessment of contamination impact in aquatic environments. Among them, the copper/zinc superoxide dismutases (Cu/Zn-SODs) are metalloenzymes which play a key role in the protection of cells in case of oxidative stress. In order to observe possible variations of an antioxidant response in relation to tidal oscillations, the copper/zinc superoxide dismutase activity (Cu/Zn-SOD) was characterized in the digestive gland and gills of blue mussels sampled at high and low shore throughout the tidal cycle. Determination of SOD activity was performed on gels after isoelectro-focusing, allowing the revelation of three isoforms. In both tissues, high-shore mussels exhibited a higher level of total SOD activity than low-shore mussels. During emersion, a decrease of total SOD activity appeared in digestive gland for both groups. In high-shore mussels, the less acidic form contributed to 75% of the total activity, the second one to 20% and the more acidic one to 5% in both tissues before air exposure. During emersion, the relative contribution of the three isoforms to the total activity was markedly changed with a significant decrease in intensity of the first isoform and parallel increases in the two other ones. After re-immersion a progressive recovery of proportions of SOD isoforms was observed. In low-shore mussels, the relative contribution of the three isoforms to the total SOD activity showed similar changes. The observed variations could correspond to changes in the redox status of the mussels during tidal oscillations.     

140 Ecophysiology of The Red Alga Porphyra Umbilicalis Kützing (Rhodophyta,Bangalies): Responses to Abiotic Stress

The effects of tidal elevation, emersion, sun exposure, and season on several antioxidant enzymes (ascorbate peroxidase, glutathione reductase, and catalase), pigments (phycoerythrin, phycocyanin, chlorophyll a and total carotene) and photosynthetic efficiency of photosystem II (Fv/Fm) in Porphyra umbilicalis were evaluated. Plants were collected monthly from sun‐exposed and shaded locations in the high, mid, and low intertidal following periods of tidal emersion ranging from 0–6 hours. Glutathione reductase activity was greatly affected by emersion during summer months, while ascorbate peroxidase and catalase activities showed no seasonal patterns. Differences in glutathione reductase and catalase levels occurred between sun‐exposed and shaded plants in the high and mid intertidal during summer. At all elevations, photosynthetic pigments showed a strong seasonal trend, with the effect of sun exposure being most apparent during summer. While total carotene increased with emersion during summer months, the combined effects of emersion and season were inconsistent for phycoerythrin, phycocyanin and chl a. Photosynthetic efficiency (Fv/Fm) decreased following emersion in summer and fall. During most months, sun exposed plants had lower Fv/Fm values compared to plants growing in the shade. This study emphasizes the importance of examining the effects of abiotic stresses simultaneously in order to reveal interactive relationships.