Patrick Reis-Santos

Ionoregulatory changes during metamorphosis and salinity exposure of juvenile sea lamprey (Petromyzon marinus L.).

SUMMARY Ammocoetes of the anadromous sea lamprey Petromyzon marinus L. spend many years in freshwater before metamorphosing and migrating to sea. Metamorphosis involves the radical transformation from a substrate-dwelling, filter feeder into a free-swimming, parasitic feeder. In the present work we examined osmoregulatory differences between ammocoetes and transformers (metamorphic juveniles), and the effects of salinity acclimation. We measured the expression of key ion-transporting proteins [Na+/K+-ATPase, vacuolar (V)-type H+-ATPase and carbonic anhydrase (CA)] as well as a number of relevant blood parameters (hematocrit, [Na+] and [Cl–]). In addition, immunofluorescence microscopy was used to identify and characterize the distributions of Na+/K+-ATPase, V-type H+-ATPase and CA immunoreactive cells in the gill. Ammocoetes did not survive in the experiments with salinities greater than 10‰, whereas survival in high salinity (⩾25–35‰) increased with increased degree of metamorphosis in transformers. Plasma [Na+] and [Cl–] of ammocoetes in freshwater was lower than transformers and increased markedly at 10‰. In transformers, plasma ions increased only at high salinity (>25‰). Branchial Na+/K+-ATPase levels were ∼ tenfold higher in transformers compared to ammocoetes and salinity did not affect expression in either group. However, branchial H+-ATPase expression showed a negative correlation with salinity in both groups. Na+/K+-ATPase immunoreactivity was strongest in transformers and associated with clusters of cells in the interlamellar spaces. H+-ATPase (B subunit) immunoreactivity was localized to epithelial cells not expressing high Na+/K+-ATPase immunoreactivity and having a similar tissue distribution as carbonic anhydrase. The results indicate that branchial Na+/K+-ATPase and salinity tolerance increase in metamorphosing lampreys, and that branchial H+-ATPase is downregulated by salinity.

Biomarker responses to environmental contamination in estuaries: A comparative multi-taxa approach

Estuaries are highly productive ecosystems subjected to numerous anthropogenic pressures with consequent environmental quality degradation. In this study, multiple biomarker responses [superoxide dismutase (SOD), catalase (CAT), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities, as well as lipid peroxidation (LPO) and DNA damage (DNAd)] were determined in two fish (Dicentrarchus labrax and Pomatoschistus microps) and four macroinvertebrate species (Carcinus maenas, Crangon crangon, Hediste diversicolor and Scrobicularia plana) from the Ria de Aveiro and Tejo estuaries over distinct months. Two sites per estuarine system were selected based on anthropogenic pressures and magnitude of environmental contamination. Antioxidant enzyme activities in fish species suggested a ubiquitous response to oxidative stress, while biotransformation and effect biomarkers exhibited higher spatial and temporal variation. In invertebrate species, biotransformation enzyme activity was clearly less variable than in fish evidencing lower xenobiotic transformation capability. Overall, largest biomarker responses were found in the most contaminated sites (Tejo), yet species-specific patterns were evident. These should be factored in multi-taxa approaches, considering that the differential functional traits of species, such as habitat use, life-stage, feeding or physiology can influence exposure routes and biomarker responses. The Integrated Biomarker Response index highlighted patterns in biomarker responses which were not immediately evident when analyzing biomarkers individually. Overall, results provided insights into the complexity of species responses to contamination in naturally varying estuarine environments. Ultimately, multi-taxa and multi-biomarker approaches provide a comprehensive and complementary view of ecosystem health, encompassing diverse forms of biological integration and exposure routes, and allow the validation of results among markers and species.

10.05 – River-Coast Connectivity, Estuarine Nursery Function and Coastal Fisheries

Due to their nature as transition systems, fishes in estuaries have affinities with both marine and freshwater faunas. Estuarine fish assemblages face physicochemical dynamics and instability, as well as human-induced changes. These systems represent a fundamental link in the life cycle of numerous species, which use them in specific life stages (in an obligatory or opportunistic manner). Many of these species represent important coastal fisheries and it is paramount to address the importance of estuaries as nurseries and their connectivity with the marine environment. Integrative approaches, as ecohydrology, may provide tools toward sustainability and restoration of estuarine ecological functions.

Ecotoxicity of the lipid-lowering drug bezafibrate on the bioenergetics and lipid metabolism of the diatom Phaeodactylum tricornutum

Pharmaceutical residues impose a new and emerging threat to the marine environment and its biota. In most countries, ecotoxicity tests are not required for all pharmaceutical residues classes and, even when mandatory, these tests are not performed using marine primary producers such as diatoms. These microalgae are among the most abundant class of primary producers in the marine realm and key players in the marine trophic web. Blood-lipid-lowering agents such as bezafibrate and its derivatives are among the most prescribed drugs and most frequently found human pharmaceuticals in aquatic environments. The present study aims to investigate the bezafibrate ecotoxicity and its effects on primary productivity and lipid metabolism, at environmentally relevant concentrations, using the model diatom Phaeodactylum tricornutum. Under controlled conditions, diatom cultures were exposed to bezafibrate at 0, 3, 6, 30 and 60 μg L-1, representing concentrations that can be found in the vicinity of discharges of wastewater treatment plants. High bezafibrate concentrations increased cell density and are suggested to promote a shift from autotrophic to mixotrophic metabolism, with diatoms using light energy generated redox potential to breakdown bezafibrate as carbon source. This was supported by an evident increase in cell density coupled with an impairment of the thylakoid electron transport and consequent photosynthetic activity reduction. In agreement, the concentrations of plastidial marker fatty acids showed negative correlations and Canonical Analysis of Principal coordinates of the relative abundances of fatty acid and photochemical data allowed the separation of controls and cells exposed to bezafibrate with high classification efficiency, namely for photochemical traits, suggesting their validity as suitable biomarkers of bezafibrate exposure. Further evaluations of the occurrence of a metabolic shift in diatoms due to exposure to bezafibrate is paramount, as ultimately it may reduce O2 generation and CO2 fixation in aquatic ecosystems with ensuing consequences for neighboring heterotrophic organisms.

Reconciling differences in natural tags to infer demographic and genetic connectivity in marine fish populations

Processes regulating population connectivity are complex, ranging from extrinsic environmental factors to intrinsic individual based features, and are a major force shaping the persistence of fish species and population responses to harvesting and environmental change. Here we developed an integrated assessment of demographic and genetic connectivity of European flounder Platichthys flesus in the northeast Atlantic (from the Norwegian to the Portuguese coast) and Baltic Sea. Specifically, we used a Bayesian infinite mixture model to infer the most likely number of natal sources of individuals based on otolith near core chemical composition. Simultaneously, we characterised genetic connectivity via microsatellite DNA markers, and evaluated how the combined use of natural tags informed individual movement and long-term population exchange rates. Individual markers provided different insights on movement, with otolith chemistry delineating Norwegian and Baltic Sea sources, whilst genetic markers showed a latitudinal pattern which distinguished southern peripheral populations along the Iberian coast. Overall, the integrated use of natural tags resulted in outcomes that were not readily anticipated by individual movement or gene flow markers alone. Our ecological and evolutionary approach provided a synergistic view on connectivity, which will be paramount to align biological and management units and safeguard species’ biocomplexity.

Screening of human and veterinary pharmaceuticals in estuarine waters: A baseline assessment for the Tejo estuary

We investigated the presence of 66 human and veterinary pharmaceuticals from seven therapeutic groups in surface waters of the Tejo estuary. Collection sites covered the entire estuary and included areas near main river inflows and wastewater treatment outfalls, traversing urban, agriculture, aquaculture, and nature reserve areas. Detection of pharmaceuticals was performed via UHPLC-TOF-MS. Pharmaceuticals were found in all sites (32 different compounds in total). Antibiotics, β-blockers, antihypertensives and anti-inflammatories were the most frequently detected (>90%), with variation in concentrations reflecting the multifaceted nature of estuarine surroundings (accumulated site contamination between 15 and 351 ng L-1). Higher concentrations of antidepressant Sertraline (304 ng L-1), non-steroidal anti-inflammatory Diclofenac (51.8 ng L-1), lipid regulator Gemfibrozil (77.0 ng L-1), antihypertensive Ibersartan (161.9 ng L-1) or antibiotic Doxycycline (128.0 ng L-1), among others, though localized may potentially impact key estuarine functions or services. Ultimately, results provide a baseline for regulatory information and future biota evaluations.

Extrinsic and intrinsic factors shape the ability of using otolith chemistry to characterize estuarine environmental histories

Reconstructing habitat use and environmental histories of fish via otolith chemistry relies on linking otolith chemical composition to the surrounding environment, as well as disentangling the consequences of ontogenetic or physiological effects that may mask environmental signals. We used multiple linear and linear mixed models to analyse the importance of environmental (temperature, salinity, water chemistry) and individual based (fish size) factors on otolith chemical composition and incorporation (Li, Mg, Mn, Sr, and Ba) of juvenile Dicentrarchus labrax, over their time within an estuarine nursery area. Multi model inference highlighted the importance of ontogeny, as well as the influence of temperature and salinity on otolith chemistry and elemental incorporation, with results indicating that intrinsic effects may potentially outweigh environmental effects. Ultimately, understanding if otolith chemistry accurately reflects fine-scale environmental variation is key to reconstruct environmental histories of juvenile fishes in estuaries and will contribute to determining the impact changing estuarine conditions may have on growth and survival.