Biomarker responsiveness in Norwegian Sea mussels, Mytilus edulis, exposed at low temperatures to aqueous fractions of crude oil alone and combined with dispersant.

Biological effects of aqueous fractions of a crude oil, alone or in combination with dispersant, were investigated in mussels, Mytilus edulis, exposed at three temperatures (5, 10 and 15 °C). Polycyclic aromatic hydrocarbons (PAHs) tissue concentrations were determined, together with genotoxicity, oxidative stress and general stress biomarkers and the Integrated Biological Response (IBR) index. The bioaccumulation of individual PAHs varied depending on the exposure temperature, with relevant bioaccumulation of phenantrene and fluoranthene at 5 °C and heavier (e.g. 5-rings) PAHs at 15 °C. The values and response profiles of each particular biomarker varied with exposure time, concentration of the oil aqueous fraction and dispersant addition, as well as with exposure temperature. Indeed, PAH bioaccumulation and biomarker responsiveness exhibited specific recognizable patterns in mussels exposed at low temperatures. Thus, genotoxicity was recorded early and transient at 5 °C and delayed but unremitting at 10-15 °C. Catalase activity presented a temperature-dependent response profile similar to the genotoxicity biomarker; however, glutathione-S-transferase responsiveness was more intricate. Lysosomal membrane stability in digestive cells decreased more markedly at 5 °C than at higher temperatures and the histological appearance of the digestive gland tissue was temperature-specific, which was interpreted as the combined effects of PAH toxicity and cold stress. It can be concluded that the profile and level of the biological effects are definitely different at low temperatures naturally occurring in the Arctic/Subarctic region (e.g. 5 °C) than at higher temperatures closer to the thermal optimum of this species (10-15 °C).

Phytoplankton community composition in relation to environmental variability in the Urdaibai estuary (SE Bay of Biscay): Microscopy and eDNA metabarcoding.

Phytoplankton monitoring is essential for the global understanding of aquatic ecosystems. The present research studies the phytoplankton community of the Urdaibai estuary, combining microscopy and eDNA metabarcoding for the first time in the area. The main aims were to describe the phytoplankton community composition in relation to the environmental conditions of the estuary, and to compare the two methods used. Diatoms Minutocellus polymorphus and Chaetoceros tenuissimus dominated the outer estuary, being replaced by Teleaulax acuta (cryptophyte), Kryptoperidinium foliaceum (dinoflagellate) and Cyclotella spp. (diatom) towards the inner area. This change was mainly prompted by salinity and nutrients. Metabarcoding revealed the presence of 223 species that were not observed by microscopy in previous studies in the estuary. However, several characteristic species (e.g., K. foliaceum) were only detected with microscopy. Additionally, microscopy covered the limitations of eDNA metabarcoding concerning quantification. Thus, to give a full insight, a combination of techniques is recommended.

Differential tissue development compromising the growth rate and physiological performances of mussel.

Differences in the food acquisition rates and in the energetic costs of metabolism seem to affect the growth rate variability of mussels. The aim of this study was to analyze if the physiological performances responsible for such growth rate variability are accompanied by structural differences at tissue or cellular level in the main organs involved in energy acquisition (gill) and processing (digestive gland). Fast growers had higher cilia density and metabolic efficiency in their gill, and well-developed digestive tissue with barely no connective tissue or atrophy. Slow-growing mussels displayed stress signs that impede the proper acquisition, digestion and absorption of food: low cilia density, low mitochondrial capacity and high antioxidant activity levels in the gills, and high atrophy of the digestive gland. The data herein explains the growth rate variability of mussels, demonstrating that morphological and functional differences exist between fast and slow growers.

Food-type may jeopardize biomarker interpretation in mussels used in aquatic toxicological experimentation.

To assess the influence of food type on biomarkers, mussels (Mytilus galloprovincialis) were maintained under laboratory conditions and fed using 4 different microalgae diets ad libitum for 1 week: (a) Isochrysis galbana; (b) Tetraselmis chuii; (c) a mixture of I. galbana and T. chuii; and (d) a commercial food (Microalgae Composed Diet, Acuinuga). Different microalgae were shown to present different distribution and fate in the midgut. I. galbana (≈4 µm Ø) readily reached digestive cells to be intracellularly digested. T. chuii (≈10 µm Ø and hardly digestible) was retained in stomach and digestive ducts for long times and extracellularly digested. Based on these findings, it appeared likely that the presence of large amounts of microalgal enzymes and metabolites might interfere with biochemical determinations of mussel's biomarkers and/or that the diet-induced alterations of mussels' digestion could modulate lysosomal and tissue-level biomarkers. To test these hypotheses, a battery of common biochemical, cytological and tissue-level biomarkers were determined in the gills (including activities of pyruvate kinase, phosphoenolpyruvate carboxykinase and cytochrome c oxidase) and the digestive gland of the mussels (including protein, lipid, free glucose and glycogen total content, lysosomal structural changes and membrane stability, intracellular accumulation of neutral lipids and lipofuscins, changes in cell type composition and epithelial thinning, as well as altered tissue integrity). The type of food was concluded to be a major factor influencing biomarkers in short-term experiments though not all the microalgae affected biomarkers and their responsiveness in the same way. T. chuii seemed to alter the nutritional status, oxidative stress and digestion processes, thus interfering with a variety of biomarkers. On the other hand, the massive presence of I. galbana within digestive cells hampered the measurement of cytochemical biomarkers and rendered less reliable the results of biochemical biomarkers (as these could be attributed to both the mussel and the microalgae). Research to optimize dietary food type, composition, regime and rations for toxicological experimentation is urgently needed. Meanwhile, a detailed description of the food type and feeding conditions should be always provided when reporting aquatic toxicological experiments with mussels, as a necessary prerequisite to compare and interpret the biological responses elicited by pollutants.

Interactive effects of osmotic stress and burrowing activity on protein metabolism and muscle capacity in the soft shell clam Mya arenaria.

Bioturbators such as sediment-dwelling marine bivalves are ecosystem engineers that enhance sediment-water exchange and benthic-pelagic coupling. In shallow coastal areas, bivalves are exposed to frequent disturbance and salinity stress that might negatively affect their activity and physiological performance; however, the mechanisms underlying these effects are not fully understood. We investigated the effects of osmotic stress (low and fluctuating salinity) and repeated burrowing on aerobic and contractile capacity of the foot muscle (assessed by the activity of succinate dehydrogenase and myosin ATPase) as well as the levels of organic osmolytes (free amino acids) and biochemical markers of protein synthesis and proteolysis in key osmoregulatory and energy storing tissues (gills and hepatopancreas, respectively) in a common bioturbator, the soft shell clam Mya arenaria. Osmotic stress and exhaustive exercise altered the foot muscle capacity of soft shell clams and had a strong impact on protein and amino acid homeostasis in tissues not directly involved in locomotion. Acclimation to constant low salinity (5 practical salinity units) depleted the whole-body free amino acid pool and affected protein synthesis but not protein breakdown in the gill. In contrast, fluctuating (5-15) salinity increased protein breakdown rate, suppressed protein synthesis, caused oxidative damage to proteins in the gill and selectively depleted whole-body glycine pool. Clams acclimated to normal salinity (15) increased the aerobic capacity of the foot muscle upon repeated burrowing, whereas acclimation to low and fluctuating salinity reduced this adaptive muscle plasticity. Under the normal and low salinity conditions, exhaustive exercise induced protein conservation pathways (indicated by suppression of protein synthesis and catabolism), but this effect was disrupted by fluctuating salinity. These findings indicate that exhaustive exercise and osmotic stress interactively affect whole-body protein homeostasis and functional capacity of the foot muscle in soft shell clams which might contribute to reduced burrowing activity of bivalve bioturbators in osmotically challenging environments such as estuaries and shallow coastal zones.

Differences in copper bioaccumulation and biological responses in three Mytilus species.

Mytilus species are important organisms in marine systems being highly abundant and widely distributed along the coast of Europe and worldwide. They are typically used in biological effects studies and have a suite of biological effects endpoints that are frequently measured and evaluated for stress effects in laboratory experiments and field monitoring programmes. Differences in bioaccumulation and biological responses of the three Mytilus species following exposure to copper (Cu) were investigated. A laboratory controlled exposure study was performed with three genetically confirmed Mytilus species; M. galloprovincialis, M. edulis and M. trossulus. Chemical bioaccumulation and biomarkers were assessed in all three Mytilus species following a 4 day and a 21 day exposure to waterborne copper concentrations (0, 10, 100 and 500µg/L). Differences in copper bioaccumulation were measured after both 4 and 21 days, which suggests some physiological differences between the species. Furthermore, differences in response for some of the biological effects endpoints were also found to occur following exposure. These differences were discussed in relation to either real physiological differences between the species or merely confounding factors relating to the species natural habitat and seasonal cycles. Overall the study demonstrated that differences in chemical bioaccumulation and biomarker responses between the Mytilus spp. occur with potential consequences for mussel exposure studies and biological effects monitoring programmes. Consequently, the study highlights the importance of identifying the correct species when using Mytilus in biological effects studies.