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09:00-09:45 Session 3: Plenary Lecture
Integrative Ecophysiology in the Anthropocene: classic approaches, current achievements & future challenges

ABSTRACT. Over the last three decades, an increasing interest in how climate change will affect life on earth has motivated many comparative physiologists to focus their research on the effects of Anthropogenic drivers (mainly represented by climate change and pollution) on the physiological performance of their study organisms in order to identify critical thresholds and tipping points for their sustainable existence or mere survival. Experimental work started with short term single driver experiments but soon developed to include multiple drivers and sometimes even successive generations in an attempt to address as much of climate change’s complexity as possible and include adaptive, trans-generational aspects to project physiological reaction norms into the future. Addressing those ‘big questions’ of high societal relevance have the merit of creating high visibility and also funding opportunities, however, classic comparative physiology and biochemistry is running the risk of becoming marginalized in this process, if the underlying physiological and biochemical processes cannot be visualized accordingly. In my talk, I will present recent examples of how integrative ecophysiology research can be used to inform society and policy, which potential pitfalls we, as a community of researchers in comparative physiology and biochemistry, should avoid and what challenges we are facing when trying to balance fundamental research with answering the ‘big questions’.

09:50-10:50 Session 2: Oral Presentations
Evaluation of the ecotoxicological impact of Cigarette Butt littering in marine and freshwater environments
PRESENTER: Matteo Oliva

ABSTRACT. Cigarette butts (CBs) represent the most littered item in the world and one of the most diffused personal waste on beaches and coastal areas. CBs can entrap a wide range of chemical compounds, such as PAHs, hydrocarbons, trace elements and nicotine, produced both by tobacco combustion and treatment during cultivation and manufacture. The aim of the present work was to evaluate the effect of CB leachates on target species with two batteries of marine and freshwater ecotoxicological bioassays. Marine tests showed an inhibition of growth with the two different algal species (P. tricornutum and D. tertiolecta), an inhibition of bioluminescence in the bacterium A. fischeri, an alteration on the correct early development of the serpulid F. enigmaticus larvae and immobilization of the copepod A. tonsa individuals at both 24 and 48 hours of exposure. Freshwater tests showed again an inhibition of the bioluminescence with A. fischeri together with a significant stimulation of radical apparatus elongation in two different superior plants (C. sativus and L. sativum), while S. saccharatum showed no statistically significant differences with controls, even if a biostimulation was again registered. Similar results were obtaind with the freshwater microalga R. subcapitata, which showed biostimulation of algal growth. Two other assays were performed with freshwater crustaceans, T. platyurus and D. magna, underlining no significant differences with controls with both species. With D. magna, also the heartbeat rate (bpm) on adults was calculated as physiological endpoint. A significant increase of the bpm in adults exposed to high concentrations of smoked CB leachate was observed after 48 h of exposure. In conclusion it was possible to define a preliminary scenario of the different effects that CB litter can cause to different key organisms in aquatic environments, underlining the importace of monitoring of this “emerging pollutant” in both internal and coastal areas.

Are Natural Deep Eutectic Solvents always a sustainable option? A bioassay-based study
PRESENTER: Matilde Sanches

ABSTRACT. The traditional use of organic solvents in various branches of industry is being rethought as these compounds very often display high volatility, toxicity and lipophilicity (related to the ability to interact with biological membranes). More recently, developments in the field of Green Chemistry are focusing on the design of more sustainable and cost-effective solvent alternatives like Ionic Liquids (ILs), bio-based solvents and natural deep eutectic solvents (NADESs). The present study aimed at performing an ecotoxicological screening of 15 NADESs using an extensive set of marine and freshwater bioassays, based on different endpoints: immobilization of the crustacean Daphnia magna, growth inhibition of Raphidocelis subcapitata and of Phaeodactylum tricornutum, larval development alterations on the serpulid Ficopomatus enigmaticus and bioluminescence inhibition of Aliivibrio fischeri. What emerged was a general absence of toxicity of all samples. However, both algal assays showed a certain degree of biostimulation, up to over 100% growth increase respect to controls with 8 out of 15 compounds tested with Raphidocelis subcapitata. Despite NADESs induced negligible toxicity effects to invertebrates, encouraging their labelling as “sustainable” solvents, the liability of their intentional or accidental release into aquatic systems may represent a serious risk in terms of ecosystem functioning impairments.

Will marine heatwaves influence the impacts of caffeine in coastal systems? Toxic effects to mussels and clams
PRESENTER: Lucia De Marchi

ABSTRACT. In the recent years, marine heatwaves (MHWs) are recognized as extreme weather events. The abrupt nature of these events can have more lethal effects on marine life than an average gradual temperature increases of seawater, severely affecting marine organism’s health, populations and ecosystem functions. Anthropogenic activities are not only responsible for climate change, but are often associated with hazardous chemicals production and release into aquatic systems, causing detrimental effects on environment and human health. Cumulative impacts resulting from anthropogenic activities have evolved in a complex scenario of multiple stressors, with a general consensus that these activities are responsible for the worldwide climate change effects observed. However, the combined effect of MHWs on the toxicity of emerging pharmaceuticals to aquatic life remains unclear. To contribute to these issues, the main goal of the present investigation was to evaluate how MHWs may increase caffeine toxicity on the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis. Bioaccumulation levels and changes on oxidative stress, metabolic capacity and neurotoxic status related biomarkers were investigated. The obtained results revealed that, despite the absence of caffeine accumulation in both species, this compound generated in both bivalves’ alteration on neurotransmission, detoxification mechanisms induction as well as cellular damage. The increase of antioxidant defence mechanisms was complemented by an increase of metabolic activity and decrease of energy reserves. These effects, moreover, were magnified in presence of a simulated MHW, suggesting to a climate-induced toxicant sensitivities’ response. On this perspective, understanding of how toxicological mechanisms interact with climate-induced stressors will provide a solid platform to improve effect assessments for both humans and wildlife.

09:50-10:50 Session 3: Oral Presentations
A multi-level assessment of the effects of Marine Heatwaves on the biology of the Manila clam Ruditapes philippinarum
PRESENTER: Luca Peruzza

ABSTRACT. Climate extreme events like Heatwaves (HWs) are a serious threat for marine ecosystems leading to massive mortality of benthic organisms and to biodiversity loss. One of the most threatened activities is bivalve aquaculture since bivalves are mostly farmed in coastal ecosystems where the effects of HWs will be more intense. However, our knowledge regarding the effects of HWs on bivalves is still limited. To fill this gap, we used the Manila clam Ruditapes philippinarum (the most farmed clam species worldwide) as model species to characterise in detail clams’ responses after an HW (i.e. 30 ˚C for 30 days). Results indicated an impairment of burying behaviour, a significant decrease in growth and hepato-somatic index and a reduction of oocyte diameter in HW-exposed clams. At biochemical level, the activity of superoxide dismutase, catalase and glutathione peroxidase were significantly increased in HW-exposed clams. The metabolic profile of clam’s foot showed higher concentrations of Alanine and Glutamate, suggesting the activation of a metabolic pathway to produce glucose via degradation of amino acids, and an homeoviscous adaptation of the cell membrane via an increase of cholesterol and a decrease of omega-6 and -3 fatty acids. Transcriptomic analysis in the hepatopancreas revealed a down-regulation of genes involved in the reproductive cycle and in the metabolism of amino acids and an upregulation of pathways like “Unfolded protein response”, Apoptosis and Necroptosis. Overall, we highlight an important trade-off in clams: despite they survived the HW, energy reserves were diverted from biological processes (e.g. reproduction, behaviour) to adjust membrane fluidity and deal with oxidative stress, protein misfolding and stress of the endoplasmatic reticulum caused by HW. Arguably clam’s resilience is pushed to its limit by HWs and if, in the field, HWs co-occur with other stressors (e.g. hypoxia), this could pose a severe threat to clam’s survival and aquaculture.

Heat waves lead to microbiome dysbiosis in marine hosts: insights from Manila clams

ABSTRACT. In the era of climate change, there is a growing recognition of the cascading effects of heat waves on the population dynamics of marine hosts’ microbiomes. In this frame, marine aquaculture is facing an increase in the prevalence of pathogenic microbes, with a consequent potentially dramatic decline in production. However, how extreme climate events shape the adaptation of marine hosts and their microbiomes, and how such processes ultimately affect aquaculture, remains poorly understood. Here, we investigated the effect of heat waves on microbiome composition in Manila clams, the third most produced bivalve species in aquaculture, and how this influences clam physiology. We characterized the microbiome in clams that were exposed to heat waves and compared it to that of a control population. Overall, thermal stress causes a significant reduction of bacterial diversity. Several bacterial genera display significant differences in relative abundance when compared to control. Specifically, we find that a distinct subset of microorganisms competes to establish a new microbiome in challenged clams. The relative abundance of the beneficial symbiont Phaeobacter decreased by 10-fold in challenged clams, while that of Vibrio spp., frequent cause of clam infections, increased by 18-fold. Notably, the microbiome of challenged clams showed higher intra-variability than control populations. Altogether, our data show that heat waves significantly affect clam microbiome by decreasing the overall bacterial richness and providing a suitable environment for pathogens to thrive. This supports the hypothesis that heat waves trigger microbiota dysbiosis in clams, and likely in all marine ecosystems. Future work is needed to establish (1) the ecological mechanisms shaping microbiomes’ response to climate change, (2) the adaptation potential of beneficial symbiotic bacteria, and (3) by what means marine aquaculture might leverage this information to prevent production losses.

Lifelong exposure to warmer temperatures does not produce higher tolerance to marine heatwaves
PRESENTER: Beatriz Pereira

ABSTRACT. The frequency and intensity of marine heatwaves have increased due to rapid climate change. However, the study of the capacity for long-term adaptation to higher temperatures has been stalled by the absence of adequate experimental systems experiencing warming across relevant timescales. Here, we examine sea urchins (Paracentrotus lividus) from S. Torpes (Portugal), a unique coastal ecosystem that maintained natural thermal fluctuations but has been warmed by 5ºC by a coal power plant for 35 years until December 2020. Sea urchins were collected from S. Torpes and Cabo Raso (control location) 9 months following the shutdown of the powerplant to determine if long-term memory of high-temperature exposure provides an advantage in dealing with marine heatwaves. Sea urchins from both locations were exposed to two laboratory-induced marine heatwaves for 15 days. Following exposure, we evaluated sea urchin oxidative stress biomarkers (HSP70, SOD, catalase, GPx, MDA), protein folding (ubiquitin), and coelomic fluid buffering capacity. Except for coelomic fluid buffering capacity, we observed that sea urchins previously exposed to higher temperatures do not retain an advantage in dealing with new heating events. This indicates that repeated exposure to warmer temperatures does not increase physiological resilience in sea urchins.

10:50-11:30Coffee Break
11:30-13:10 Session 1: Oral Presentations
Lipidomic analysis of mussel hemocytes exposed to polystyrene nanoplastics

ABSTRACT. Plastic production and usage has exponentially increased in the last decades around the world. Due to the insufficient waste management, a significant amount of plastic ends up in the environment, where they tend to fragment into micro- and nano-plastics (NPs), and accumulate in aquatic organisms with still unknown effects. Although studies have indicated that lipid metabolism is a main target of NPs, this mechanism has not been extensively explored. In this study, we evaluated changes in the lipidome of mussel hemocytes after exposure to polystyrene (PS) NPs of 50 and 500 nm, at two different concentrations (10x6 and 10x9 particles/mL) for 24 h. The lipidome of hemocytes, analyzed by FIA-ESI (+/-) Orbitrap, was characterized by a relatively high abundance of cholesteryl esters (CEs) and phosphatidylcholine-plasmalogens (PC-Os/PC-Ps), involved in cell's defense against oxidative stress and membrane reorganization. In hemocytes exposed to PS NPs, a number of highly unsaturated membrane lipids were down-regulated, indicating a reorganization of the cell membranes after exposure to the particles and an oxidation of lipids with a high number of double bonds. This decrease was more evident as the number of particles increased and the particle size decreased. The analysis of culture medium suggested increased release of vesicles enriched in triglycerides (TGs) from hemocytes. It remains to be explored how these subtle changes in the lipidome of mussel´s hemocytes induced by NP exposure can be translated into potential impacts on the immune function and host/pathogen interactions of these key marine organisms. *Work funded by grants to the consolidated group IT1302-19 and IT1743-22 (Basque Government), project NACE CTM2016-81130-R (MINECO), CAS6 project (EU JRC CAS 30602) and project PGC2018-097513-B-I00 (MCIU, AEI and FEDER).

Fate and effects of a reduced graphene oxide-silver nanoparticle hybrid material on mussels Mytilus galloprovincialis

ABSTRACT. Research on graphene family nanomaterials (GFNs) has bloomed in recent years. Currently, a new generation of hybrid nanomaterials that combine GFNs with silver nanoparticles (Ag NPs) has received increased attention due to their enhanced properties. But, the concern about their possible toxicity is also rising because Ag NPs are known to be toxic for several organisms and many of the applications of these novel nanocomposites involve their direct application in the marine environment. This work aims to assess the potential hazards that a composite of reduced graphene oxide (rGO) with Ag NPs (rGO-Ag) could pose towards marine mussels. Mussels were exposed for 21 days to 500 µg/L of rGO-Ag and to the equivalent concentration of rGO (375 µg/L) and Ag NPs (125 µg/L) alone for comparison. rGO nanoplatelets were detected by Raman spectroscopy in the lumen of the digestive tract and feces of exposed mussels. Mussels exposed to rGO-Ag and Ag NPs accumulated Ag in their tissues. Exposure to rGO-Ag induced the activity of AchE, catalase activity and oxidative damage at day 3 of exposure and increased the prevalence of brown cell aggregations in gonads. Both rGO and rGO-Ag caused inflammatory responses in gonads and decreased the digestive gland index after 21 days of exposure. Further, rGO caused transitory DNA damage nd inhibited catalase activity. Exposure to Ag NPs at the equivalent concentration as in the rGO-Ag hybrid lead to multiple alterations including downregulation of catalase and hsp90 transcription levels, DNA damage and altered enzyme activities related to aerobic and anaerobic metabolism, neurotransmission and phase 2 biotransformation. In addition, 21 days of exposure to Ag NPs caused inflammatory responses in gonads and mortality of few individuals. In conclusion, both rGO and rGO-Ag were able to cause deleterious effects to mussels and Ag NPs exerted less toxicity when dosed in the form of rGO-Ag than alone.

Ecosafety assessment of nanocellulose-based sponges on sea urchin reproduction

ABSTRACT. Nanocellulose-based sponges (CNS) are a promising eco-friendly and sustainable engineered nano-structured material for seawater nanoremediation developed following an eco-design approach [1]. Even though CNS have been proved to be highly efficient in seawater decontamination from metals, any potential environmental risk associated with their in situ applications needs a broader ecological assessment [2, 3, 4]. Therefore, spermiotoxicity and embryotoxicity bioassays were performed upon exposure of male gametes and embryos of two Mediterranean sea urchin species, i.e. Paracentrotus lividus and Arbacia lixula, to CNS. The suitability of these tests for the risk assessment of ENMs was evaluated and the eligibility of the A. lixula as alternative species in ecotoxicity testing has been investigated by developing appropriate bioassays. Female gamete fertilization competence and gamete quality were also studied by developing new bioassays and estimating multiple physiological parameters. Regardless of the species tested, CNS showed effects on: (i) sea urchin gamete physiology altering mitochondrial activity and the oxidative status; (ii) sperm fertilizing ability; (iii) egg fertilisation competence; and (iv) embryo development inducing a grow delay and abnormal plutei. The observed ecotoxicity resulted from the release of chemical additives used during their synthesis; hence it was reduced upon several conditioning steps in which CNS were washed using natural seawater. Overall, the obtained results indicated that the two sea urchin species have a similar CNS sensitivity supporting the use of A. lixula in the ecotoxicity tests and that standardised bioassays with P. lividus are sensitive and valuable tools for the ecosafety assessment of ENMs for water remediation and useful for achieving an ecosafe design.

Innocent Until Proven Guilty: Chronic Toxicity of AgNPcitLcys for Water remediation Reveals the Need to Reconsider Ecosafety Testing

ABSTRACT. Silver nanoparticles (AgNPs) are widely employed in everyday consumer products as biocides but can be also applied as sensors, adsorbents and photocatalysts for water pollution detection and remediation. However, since the ecotoxicity of nanosilver still pose limitations to its environmental application their production and application should rely on safety assessment for possible human and environmental health implications by adopting a more ecological exposure testing strategy. Here, we tested acute and chronic toxicity of novel AgNPs coated with citrate and L-cysteine (AgNPcitLcys) synthesized as sensor and adsorbent of Hg2+ in polluted waters. AgNPcitLcys behavior and release of Ag ions were monitored in exposure media by dynamic light scattering (DLS) and inductively coupled plasma-mass spectrometry (ICP-MS). Freshwater and marine microalgae and microcrustaceans were exposed to a range of AgNPcitLcys concentrations (1 µg/L–100 mg/L) in order to resemble realistic exposure scenarios and establish an effect threshold. Acute toxicity to the microalgae Raphidocelis subcapitata and Phaeodactylum tricornutum (1-1000 µg/L) were assessed while both acute and chronic toxicity to the microcrustaceans Ceriodaphnia dubia (1-100 µg/L) and Artemia franciscana (0.1-100 mg/L) were investigated. AgNPcitLcys caused low or no acute toxicity to both microalgae (EC50>1000 µg/L) and microcrustaceans, while chronic exposure (7-14 days) revealed severe effects in both Ceriodaphnia dubia and Artemia franciscana (EC50, respectively, of 24 µg/L and 5.087 mg/L). Based on the measured low concentrations of dissolved Ag in exposure media, higher toxicity than expected was observed, in all investigated species, suggesting the occurrence of a nano-related effect. Our findings highlight the need to focus more on chronic rather than only acute toxicity, which can underestimate the risk posed by AgNPs, in order to promote their safety for environmental applications (ecosafety).

Unravelling the mechanism of toxicity of polystyrene nanoparticles (PS NP) amino-modified (PS-NH2) during the embryogenesis of the chordate Ciona robusta: the development of an Adverse Outcome Pathway

ABSTRACT. The growing concern around nanoplastics (NPs) has led to a wide investigation of their effects on marine life. Nowadays, there is an increasing challenge to understand their mechanism of action for calculating and predicting the toxicological effects at organism and population levels. Here, we evaluated the effects of polystyrene nanoparticles (PS NP) as proxy for nanoplastic, of 50 nm amino-modified (PS-NH2) during the embryogenesis of the ascidian Ciona robusta at transcriptomic level, considering also the potential chemicals released by PS-NH2, for the development of a putative Adverse Outcome Pathway (AOP). Several genes and pathways involved in stress response, energy metabolism resulted affected by 10 and 15 μg mL-1 to PS-NH2 after 13 h of incubation. Moreover, chemical analysis showed the release of byproducts such as styrene monomers and diphenyl sulfone in the exposure media, which could have influenced the expression of some factors involved in receptor pathways (TGF-β and PI3K-Akt signaling) and genes coding for different extracellular matrix proteins (collagen, thrombospondin, integrin, and laminin). Based on these results, the putative AOP for PS-NH2 included oxidative stress, the interaction between plastics byproducts and TGF-beta signaling, and hypoxia as the Key Events (KE), resulting in an altered locomotor activity, impaired development and decline of population as final Adverse Outcome (AO). This study provides new insights into the understanding of PS-NH2 toxicity on aquatic organisms and could potentially be used for environmental risk assessment purposes.

11:30-13:10 Session 2: Oral Presentations
Effects of 4-MBC environmental pollution on gametes and adults of the mussel Mytilus galloprovincialis in a multiple stressor context
PRESENTER: Alessia Cuccaro

ABSTRACT. In marine-coastal systems, organisms are exposed to a growing number of stressors, with associated ecotoxicological risks at different ecological levels. Among stressors, changes in environmental abiotic factors related to Climate Change (CC) (i.e,. salinity shifts) or new pollutants (i.e., cosmetic ingredients) have been identified as key emerging concerns. Particularly, 4-methylbenzylidenecamphor (4-MBC), as one of the most used UV-filters in several products, tends to be continuously discharged into aquatic environments, posing high risk for inhabiting organisms. However, the toxicity exerted in non-target species at different stages is almost unknown, especially under predicted CC scenarios. The present study aimed to clarify if and how salinity variations influenced the impacts of 4-MBC environmental contamination on both male gametes and adults of M. galloprovincialis. By in vivo and in vitro approaches, physiological and biochemical parameters were evaluated in short-/long-term assays, providing ecologically more relevant information on organism responses. Results showed that combined stressors (4-MBC and salinity changes) induced redox status imbalance and oxidative stress in both stages, with ecotoxicological effects in terms of: I) sperm structural impairments, motility and kinetic alterations, DNA damage and shorted longevity; II) physiological dysfunctions, metabolic and energy reserve impairments, DNA damage as well as oxidative and biotransformation enzyme activity variations in adults. A salinity-dependent uptake of waterborne 4-MBC was also detected after 28-days exposure. Additive effects resulted evident, pointing out salinities 20 and 40 as worst exposure conditions for both sperms and adults, since caused major toxic impacts. By the combination of short/long-term exposure and multi-biomarker integrated approach, this study provides evidence that CC/pollutant scenarios may impair mussels’ populations, affecting reproduction success and growth.

Effects of environmental concentrations of the fragrance Amyl salicylate on the Mediterranean mussel M. galloprovincialis
PRESENTER: Massimo Milan

ABSTRACT. Amyl salicylate is a fragrance massively used as a Personal Care Product and following the discharged in wastewaters may end up in the aquatic environment representing a potential threat for the ecosystem and living organisms. Recently, high Amyl salicylate concentrations were detected in water of the Venice Lagoon, a vulnerable area continuously subjected to the income of anthropogenic chemicals. However, no studies investigated the possible consequences of AS exposures on species inhabiting this ecosystem. Here, we applied a multidisciplinary approach to investigate the possible effects of the fragrance Amyl salicylate on Mediterranean mussels. To reach such a goal, bioaccumulation, cellular, biochemical, and molecular analyses (RNA-seq and microbiota characterization) were performed in tissues of mussels exposed for 7 and 14 days to different AS Venice lagoon environmental concentrations (0.1 and 0.5 µg L-1). Despite chemical investigations suggested low Amyl salicylate bioaccumulation capability, cellular and molecular analyses highlighted the disruption of several key cellular processes after the prolonged exposures to the highest Amyl salicylate concentration. Among them, potential immunotoxicity and changes in transcriptional regulation of pathways involved in energy metabolism, stress response, apoptosis and cell death regulations have been observed. Conversely, exposure to the lowest Amyl salicylate concentration demonstrated weak transcriptional changes and transient increased representation of opportunistic pathogens, as Arcobacter genus and Vibrio aestuarianus. Overall, this study provides the first overview on the effects of Amyl salicylate on one of the most widely farmed mollusk species.

Motility of Mytilus galloprovincialis hemocytes after in vitro exposure to paracetamol

ABSTRACT. Pharmaceuticals released into the environment represent an environmental problem of increasing concern for the health of ecosystems and humans. The present work aimed to study the effect of paracetamol, one of the most widely used anti-inflammatory drugs, on the hemocyte motility in the bioindicator organism Mytilus galloprovincialis. Motility is an intrinsic characteristic of hemocytes since the first phase of their immune response is represented by their recruitment and migration to the site of infection. We developed a cell motility assay on hemocytes adhering to the bottom of a 96-well black polystyrene microplate maintained at 15°C. Cell migration and velocities were quantified by cell tracking under timelapse microscopy imaging at different exposure times from 1h to 48h. Cells were labeled with the fluorescent probes Lysosensor green and Hoechst to visualize lysosomes and nucleus respectively. In parallel, cell vitality was assessed by the propidium iodide dye exclusion assay. Cell velocity showed to be cell type dependent. Granulocytes, which showed positive staining with the acidotropic probe Lysosensor green, exhibited the highest migration rate. After 24 h of culture, migration velocities transiently increased in control cells and then declined progressively to reach the initial value after 48h. Cells exposed to 200µg/l paracetamol, a concentration widely found in aquatic environments, showed a slight stimulation of cell migration after 1h of incubation, while they failed to exhibit the typical transient stimulation of cell motility after 24h observed in control cells. These results suggest a multitarget effect of the drug on cell motility at different exposure times. In conclusion, the obtained results demonstrated hemocyte motility to be a cellular function very sensitive to paracetamol exposure and they provide a novel approach to assessing the effect of environmental chemical pollutants on hemocyte activity in environmental biomonitoring.

Molecular modeling and docking simulation with fish beta2 adrenergic receptors and the beta-blocker propranolol: new insights to disclose molecular initiating events in Adverse Outcome Pathway and risk assessment of emerging pollutants

ABSTRACT. EU pharmacovigilance legislation recognizes that pollution of aquatic ecosystems from pharmaceuticals is an emerging environmental issue, and addresses the need to improve environmental monitoring through analytical and complementary (biological in vitro and in silico testing) techniques. Besides the evaluation of specific toxicological endpoints, approaches assessing perturbation of whole biological pathways are considered for pharmaceutical environmental risk assessment. The Adverse Outcome Pathway (AOP) describes a sequence of events commencing with the initial interaction of a stressor with a biomolecule that causes a perturbation in animal biology, progressing through intermediate key events up to an apical adverse outcome relevant to risk assessment or regulatory decision-making. The AOP initial anchor is the molecular initiating event (MIE), the first interaction of the stressor with a biological system, at the molecular level. To investigate a potential MIE, in this study homology modeling predicted the 3D structure of β2 adrenergic receptors (β2-AR) from four teleost fish: the Atlantic salmon (Salmo salar), the Brown trout (Salmo trutta), the Rainbow trout (Oncorhynchus mykiss), the Atlantic herring (Clupea harengus). Molecular docking simulated the interactions with propranolol, a model β-blocker highly prescribed to treat blood hypertension and cardiac arrhythmias, and a widespread pollutant in aquatic systems. This docking analysis shows that residues involved in the ligand-binding pocket are highly conserved between fish and mammals, but orientation of some key residues in fish structures differ from the human model. Conformation of propranolol docked into the fish β2-ARs provides more hydrophobic interactions promoting ligand-binding stabilization. These results attempt contributing to understand fish β-AR functions and to give new information for AOP analysis of β-blockers and other aquatic contaminants sharing structural similarity with AR ligands.

Combined effects of anticancer drugs in Danio rerio
PRESENTER: Daniel Bruno

ABSTRACT. Pharmaceuticals are considered emerging contaminants and concerns about their environmental impact have been raised in the last decades, due to their detection in the environment. One group of pharmaceuticals commonly found in the environment are anticancer agents (AAs). AAs are one of the least studied groups with respect to environmental impact and may present specific risks to aquatic species. Unfortunately, under a real scenario, AAs do not exist alone in the environment but rather in mixtures with other AAs and/or chemical substances simultaneously. It is thus urgent to assess the effects of eventual mixtures to a non-target aquatic species. Aiming at understanding the potential hazard of AAs mixtures, the single and joint effects of two AAs (trabectedin and doxorubicin) were evaluated in zebrafish embryos. The OECD 236 Fish Embryo Acute Toxicity (FET) Test was used to determine acute toxicity of the two anticancer drugs on embryos and larvae of the zebrafish, Danio rerio. Briefly, fertilized eggs were exposed initially to a series of concentrations of each AA for 96h. Once the LC50 values were determined for each AA, they were transformed into Toxic Units (TU) and a full factorial design was used to predict their joint toxicity pattern. Embryos were exposed during 96h to a series of combinations of the two AAs along with control treatments. The MIXTOX model was used to determine joint toxicity effects, starting on the potential additivity of effects or any further deviation for synergism or antagonism. The single acute toxicity tests showed that trabectedin and doxorubicin caused mortality and several malformations in zebrafish embryos/larvae, such as pericardial edema, yolk sac absorption and tail deformities. LC50 values for trabectedin and doxorubicin at 96h of exposure were to 43 µg/L and 40 mg/L, respectively. Preliminary results of the mixture exposure indicate interaction between the tested chemicals.

13:10-14:40Lunch Break
14:40-16:00 Session 2: Oral Presentations
Comparison of two studies (environmental / experimental) on the potential effects of sacrificial anodes on the health status of the black scallop Mimachlamys varia.
PRESENTER: Marine Barbarin

ABSTRACT. Coastal areas are generally places of high economic activity, linked to the presence of port areas. These port areas have developed marine ecosystems in their basins, which may be subject to various forms of contamination resulting from their activities, including heavy metals. Indeed, they can come from roads, river discharges, careening stations, but also from cathodic protection equipment. To date, few biomonitoring studies have examined the potential impact of cathodic protection on biodiversity in port areas. This phenomenon slows down the corrosion of steel structures caused by seawater thanks to sacrificial anodes, in most cases composed of aluminium, zinc and indium. Large quantities of anodes are needed to protect submerged infrastructure, and therefore the amount of corrosion products resulting from anode degradation appears to be considerable. Studies were therefore conducted in 2020 and 2021, both in port and in the laboratory, to observe whether sacrificial anodes have an impact on the health status of black scallops Mimachlamys varia through a multi-biomarker approach. Described in the literature as a sentinel species, the scallop is a sedentary filter feeder with the ability to bioaccumulate and tolerate large quantities of contaminants in its body. A first study was set up in the seaport and marina of La Rochelle, on several sites, in order to observe the potential effect of anodes through an experimental approach. A second study, carried out in the laboratory, aimed to reproduce the same conditions as the first study with regard to anodes, but without the influence of port activities. The first results showed that in a port area the different activities (trade, careening) and the weather conditions greatly influence the data and that the potential effect of sacrificial anodes is not visible. The laboratory results showed little effect of sacrificial anodes on individuals, except under extreme conditions.

First study on the determination of baseline biomarkers in Mimachlamys varia for an intra-port environmental biomonitoring in French Atlantic coastline (La Rochelle)
PRESENTER: Marine Barbarin

ABSTRACT. The characterization of organic and inorganic environmental pollution in coastal ecosystems, such as port areas, is complex and difficult to carry out due to the effect of environmental variables, as well as anthropic activities, for the evaluation of the monitoring of the environments quality. The objective of this work was to define a statistical method, taking into account the influence of confounding factors likely to induce variations in the health status indicators studied in the black scallop (Mimachlamys varia), which would make it possible to define reference values for the biomarkers. This study thus made it possible to describe, for five biomarkers, reference data for individuals placed on sites more or less strongly impacted by environmental contamination specific to port areas for more than two years. The methodology used took into account abiotic parameters such as temperature and salinity, as well as the biometry of the individuals and the location of the study sites with the fuel station or the marina careening station for example. In this context, reference values for the biomarkers SOD (SuperOxyde Dismutase), GST (Glutathion-S-Tranferase), MDA (MalonDiAldehyde), AChE (AcetylCholinEsterase) and LAC (LACcase) for this marine species could be established. All of these results made it possible to calibrate and validate our approach in terms of active biomonitoring for the evaluation of a good chemical and ecological status of the environment of a port located on the French Atlantic coast. This method will be used again for other bivalve species, the Crassostrea gigas oyster and the Mytilus edulis mussel.

Antioxidant responses induced by PFAS environmental contamination in a freshwater fish species of the Veneto region
PRESENTER: Elisabetta Piva

ABSTRACT. Due to the toxic effects generated by per- and poly- fluoroalkyl substances (PFAS) in humans, as well as in other living organisms, the interest towards this new class of chemical compounds has grown exponentially across the world in recent decades. However, information about possible antistress responses in organisms during chemical exposure is currently insufficient and mostly linked to human-related studies. The present work aims to evaluate possible physiological responses induced by PFAS in a freshwater fish species of the Veneto Region. The sampling of the target species was performed nearby the city of Vicenza, a geographical area known to be involved in one of the most serious PFAS contamination events. Squalius cephalus, a potamodromous species, was collected from three rivers with different levels of PFAS contamination. Several oxidative stress indicators have been tested and the results indicated that liver is one of the target organs of PFAS toxicity. In fact, it is possible to see a slightly correlation between PFAS accumulation in this organ and lipid peroxidation, a consequence of ROS damage at the level of cell membranes. In this organ, the antioxidant system appears to be responsive against the oxidative stress risk. In particular, the analysis of gene expression of mitochondrial antioxidant enzymes highlighted the induction of sod2. (Supported by the European Union Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie Actions, Grant Agreement No. 860800).

Environmental health assessment of the northwest Portuguese coast – biochemical biomarker responses in the marine gastropod Phorcus lineata
PRESENTER: Dércia Santos

ABSTRACT. Coastal areas are frequently impacted by anthropogenic pollution, due to intensely human activity in these zones. In turn, it has significant implications for the ecological quality and economic management of these ecosystems. Our study aimed to monitor the impacts of anthropogenic pollution in four Portuguese locations on the northwest coast and identify the most affected areas and/or seasons by applying a multi-biomarker approach. Specimens of Phorcus lineata were collected in four sites with different anthropogenic pressures along the northwest Portuguese coast (1. Amorosa; 2. Cabo do Mundo; 3. Homem do Leme; 4. S. Félix da Marinha), at two seasons – summer and winter. The specimens were sampled on the rocky shore during low tide and transported to the laboratory, where were sacrificed and frozen (−80 °C) until further analysis. Several biochemical biomarkers, including reactive oxygen species, protein carbonyl content, lipid peroxidation (LPO), carboxylesterase (CE) and antioxidant (superoxide dismutase – SOD, catalase – CAT, glutathione S-transferase – GST) and neurotoxicity (acetylcholinesterase – AChE) enzymes were measured. The Integrated Biological Index (IBR) was used to assess the toxicological status of this species. The results showed seasonal variation, with the ROS, LPO, and CE and GST activities depending particularly on the season, but SOD and CAT activities being similar between summer and winter. CAT showed lower activity in site 1 than in the other sites, in both seasons (p<0.05). The IBR index showed that biomarker responses were higher in winter. The gastropods of local 3 showed the highest IBR value at the winter season, indicating that are under higher stress, and with the LPO biomarker being the most discriminant factor in this location. Overall, this study shows that P. lineata can be a suitable bioindicator species for environmental biomonitoring and that the IBR index allows identifying temporal patterns of contamination.

14:40-16:00 Session 3: Oral Presentations
Effect of Ocean Acidification on the production of infochemicals and other important metabolites by the benthic diatom Cocconeis neothumensis in Posidonia oceanica meadows
PRESENTER: Emanuele Somma

ABSTRACT. Posidonia oceanica produces a complex ecosystem and associated plant and animal communities are stabilized by complex chemical and trophic relationships. Diatoms of the genus Cocconeis are known for their key role in the production of apoptogenic compounds influencing the sex reversal of the shrimp Hippolyte inermis and these relationships are vital to assure stability to the shrimp populations. Here we have collected diatoms in control meadows at normal pH and also in test meadows at pH 7.6, close to CO2 vents in the bay of Naples. Diatom strains (Cocconeis neothumensis) have been isolated, taxonomically identified under SEM microscopy and cultured. Shrimp post-larvae have been cultured and fed on diatoms collected at normal pH or, alternatively, with diatoms isolated in acidified sites. After 5 days of feeding post-larvae have been fixed in RNA-later and their RNA has been extracted to be submitted to reat-time investigations on seventeen key genes known to be modified by the ingestion of these diatoms. The results demonstrate that the two strains collected in areas at different pH are characterized by significant differences in the production of secondary metabolites and that ocean acidification may drastically impact the physiology and the ecology of plant and animal species co-evolved in acidified conditions.

Combined effects of ocean acidification and copper sulfate on larvae of Arbacia lixula
PRESENTER: Matteo Nannini

ABSTRACT. CO2 emissions are considered one of the main factors contributing to global climate change (GCC). The challenge of research on GCC at sea is to determine species capacity for adaptation to future drivers, such as Ocean Acidification (OA). Vulnerability of organisms to predictable environmental changes can be mitigated by parental anticipatory buffer effects via acclimatization or adaptation to stressful environments. However, these responses may be too slow for future GCC, thus impairing the ability of local populations to survive. Natural phenomena reproducing future scenarios of OA (CO2 vents) can be used to study the adaptation potential of natural populations to GCC. Furthermore, susceptibility to other environmental stressors, such as heavy metals, may be also altered under acidified conditions, resulting in more detrimental effects in exposed organisms. Therefore, the combined effects of different levels of pH (8.1 and 7.7) and different copper sulfate concentrations (0 mg/L; 5 mg/L; 20 mg/L) were investigated in the sea urchin Arbacia lixula larval development. Adult sea urchins were collected in 2 sites characterized by different pH levels, San Pietro (pH 8.1) and Castello Aragonese (pH 7.7, due to the presence of CO2 vents) to obtain 2 different experimental larval populations to be exposed to different experimental conditions (pH level*Cu concentration) for 48 hours post fertilization. Our results highlighted a different pattern of response between the two experimental larval populations. Normal larval development was significantly affected by parental population*pH*[Cu], with an increased percentage of morphological abnormalities in larvae from adults living in ambient pH conditions, suggesting that i) pre-exposure of A. lixula adults to OA might trigger a better response against heavy metals in their off-springs or that ii) reduced pH might have chemically altered copper sulfate transforming it in a lesser toxic form.

Surviving in a changing ocean. Investigations on ocean acidification tolerance in different invertebrates from the CO2 vents of Ischia Island (Italy), from cellular pathways to susceptibility to pollution

ABSTRACT. Ocean acidification (OA) is one of the most relevant environmental threat to ocean biodiversity. Understanding how organisms cope with low pH is extremely important to foster conservation of marine ecosystems under such climate-related stressor. In this context, the study of populations living under naturally acidified conditions - such as those occurring in CO2 vent systems - can provide essential information to understand resistance and resilience to OA, and so to make comprehensive predictions of ecological processes which can occurr in future acidified oceans. The goal of our investigations was to identify the molecular and physiological mechanisms underlying the adaptive response to OA in different invertebrates living in the CO2 vent systems of Ischia Island. The polychaetes Platynereis spp. and Syllis spp., and two calcifying mollusks Mytilus galloprovincialis and Patella spp. were selected according to their different ability to persist in the low pH zones of the vents, likely related to a different eco-physiology and acclimatization/adaptation to OA. First we investigated modulation of the antyoxidant system and of some metabolic endpoints. Furthermore, we investigated the responsiveness of populations living inside the vents compared to those of control zones (normal pH conditions) to face exposure to acetone and copper. This approach aimed to evaluate if organisms able to cope with acidified conditions have also an enhanced resistance against pollution, or whether physiological modifications, induced to cope with the acidified conditions, will reduce the capacity of the organism to respond to chemical contamination and further challenge their health and ability to survive. Our study broadens the current knowledge on the mechanisms that might promote tolerance to OA in marine invertebrates. This is essential for predicting the vulnerability of organisms to face multiple environmental challenges occurring in the future oceans of the Anthropocene.

The ABC response of Halomicronema metazoicum (Cyanobacteria) to ocean acidification scenarios
PRESENTER: Silvia Simonetti

ABSTRACT. Increased fossil fuel combustion has led to higher atmospheric carbon dioxide (CO2) concentration by 30% producing a rise in CO2 ocean absorption and a consequent fall in surface pH by near 0.1 units. This phenomenon, also called ocean acidification (OA), can severely affect marine life due to the physiological stress from low-pH/high-pCO2. However, certain marine species have notable abilities to deal with and tolerate naturally acidified conditions. Recent findings on Cyanobacteria species have marked the involvement of ATP-binding cassette (ABC) transporters in acid stress responses. Researchers have suggested that survival in acidified environments of studied bacteria can be ascribed to transport of substances facilitating the acid stress tolerance by the ABC proteins. The marine cyanobacterium Halomicronema metazoicum has been isolated from Posidonia oceanica leaves in meadows also exposed to low-pH conditions and it is considered able to tolerate such acidified conditions. This feature makes it an interesting organism to evaluate molecular mechanisms at the basis of resistance to low-pH environments. Hence, Halomicronema mattes from cultures maintained within Ischia Marine Centre (Italy) have been exposed to 3 different low-pH conditions (7.7, 7.2, 6.5) for 7 days in order to provide a first assessment of the involvement of ABC transporters in tolerance to OA scenarios through ABC-like slr2019 gene expression analysis. Results have confirmed that Halomicronema can tolerate low-pH environments since it has grown during the 7-days exposition. Upregulation of slr2019 gene has been detected at predicted-pH by 2100 (7.7) while no gene modulation has been found at lower pH (6.5). This suggests an hormesis response to decreasing pH environments and further investigation will be needed to confirm this hypothesis. Unravel the role of ABC in acid stress resistance could be a first step to highlight cellular and molecular mechanisms behind adaptive responses to OA.

16:00-17:00 Session 2: Oral Presentations
Integrated assessment of the health status of mussels Mytilus galloprovincialis in the Bay of Biscay
PRESENTER: Sonia M. Landro

ABSTRACT. Integrated monitoring approaches combining the measurement of pollutants and biomarker responses at different levels of biological organisation have been widely used to study the health status of marine ecosystems. The main objectives of this work are 1) to assess levels of trace metals, total PAHs, and total PCBs in mussels and sediment and 2) to study cell and tissue biomarkers in Mytilus galloprovincialis collected in June 2021 in Plentzia (reference site) and the leisure harbour of Arriluze (polluted site). In sediment, total PAH values were higher than the limit of detection (LOD) in both sites whereas total PCB was higher than the LOD only in Arriluze. Contrary to previous results, in mussels, total PAH and PCB content were under the LOD in both sites. In agreement, there were no significant differences between sites in the peroxisomal acyl CoA oxidase activity. The content of all metals in sediments was much higher in Arriluze than in Plentzia but in mussels, only Cd and Pb were higher in Arriluze. The Comet assay revealed that the levels of DNA strand breaks were significantly higher in Arriluze than in Plentzia while there were no differences between sites for micronuclei frequency although the frequency of nuclear buds was higher in Arriluze. In agreement, the volume density of basophilic cells was significantly higher in mussels from Arriluze. The digestive tubule atrophy was prominent in Plentzia, although the frequency of histopathologies such as haemocytic infiltration, granulocytomas, fibrosis, and necrosis (amongst others) was higher in Arriluze. The sex ratio in Arriluze was 1F:3M, which might indicate an endocrine disruption effect that will be further studied. Finally, the condition index was higher in Arriluze, possibly because of its higher organic matter content compared to Plentzia. Overall, these results are relevant to understanding the long-term trends of legacy pollutants and the health status of sentinel mussels in the area.

Effects of heavy metals exposure on microalgae from the Gulf of Naples, proposed as a candidate for bioremediation strategies
PRESENTER: Elena Cavalletti

ABSTRACT. Heavy metals (HMs) are used in a wide range of applications and consequently discharged into the environment, where they constitute a growing threat for organisms and ecosystems, due to their long persistence and their potential accumulation in living organisms. Microalgae have been shown to remove HMs from aquatic environments. Microalgae had to design strategies for their uptake and homeostasis and, in some cases, are even able to compartmentalize them into vesicles and/or organelles. In addition, they can show visible markers of HM stress (cell and organelles size/shape alterations, impairment in growth and photosynthesis), and have thus also been proposed as bioindicators. Hence, reliable, easy-to-apply, high throughput optical techniques are needed for the investigation of HMs effects on microalgal cell and organelles morphology. In this study we conducted tolerance experiments for the essential metal Copper (Cu) and the non-essential metal Cadmium (Cd) on a strain of Skeletonema pseudocostatum isolated from the Sarno River mouth (Gulf of Naples, Italy). Our results show that this strain is resistant to environmentally relevant concentrations of toxicant and able to sustain a positive growth up to ca. 10 µM, removing part of the metal in solution, even though levels of activity of ROS-scavenging enzymes, such as catalase and ascorbate peroxidase, seem to indicate that at high (25 µM) metal concentrations oxidative stress is induced in the cells. These results suggest that S. pseudocostatum may be a suitable candidate for HMs bioremediation. Moreover, we here propose quantitative phase microscopy (QPM) optical techniques for the rapid analysis of HMs effects on S. pseudocostatum. Our preliminary QPM experiments show that these label-free techniques are really sensitive to cells small morphological variations due to HM exposure, even at sub-lethal doses, and could thus help in paving the way for the use of this species as potential bioindicator of HM stress.

First identification and heterologous expression of microalgal hydrolases for phthalates degradation

ABSTRACT. Phthalate esters (PEs or simply phthalates) were introduced as plasticizers in 1920, and contributed to the spread of plastic worldwide. Given PE tendency to leach from the host matrix and migrate into their surroundings, they are easily absorbed by human body, causing health detriments in reproductive, cardiovascular, respiratory and nervous systems. Given their classification as endocrine disrupting chemicals (EDCs), many bacterial genomes and metagenomes have been screened for enzymes capable to remove PEs from the environment with a bioremediation approach.

With recent advances in microalgal genomics and transcriptomics, it has become easier to identify sequences encoding enzymes with biotechnological applications. Given that the marine diatom Cylindrotheca closterium has shown bioremediation potential against PEs, our work has focused on the discovery and characterization of the enzymes involved in this relevant process.

Here we present the first identification of two putative PEs hydrolases (provisionally named CcDBPH1 and CcDBPH2) in microalgae. Docking simulations and RT-qPCR expression analysis were employed to describe the interaction between the identified proteins and the expected substrate. Obtained results confirmed theorized CcDBPH1-2 role in PEs degradation.

The sequences were therefore expressed in heterologous systems and the esterase and PE-degradation activity of the produced proteins was tested with positive results, corroborating their bioremediation potential. Potential PEs hydrolases found also in other microalgal transcriptomes highlights the great potential of microalgae in PEs degradation strategies, previously pertaining almost exclusively to bacteria. Taken together, our evidences open to an increased understanding of the chemical and molecular processes underlying microalgal response to plastic pollutants, that will help us to better define adaptation strategies employed by microalgae and how they can benefit human life.

16:00-17:00 Session 3: Oral Presentations
Differing life histories affect cellular, biochemical and physiological responses of sea urchins to ocean acidification: Paracentrotus lividus as a case study.
PRESENTER: Davide Asnicar

ABSTRACT. Ocean acidification affects in many ways marine organisms. The alteration of the seawater chemistry may lead to physiological, immunological and antioxidant defence alterations. Nonetheless, different environmental conditions experienced throughout the animal life at the residence site may influence the susceptibility to seawater acidification. Here, Paracentrotus lividus specimens from the Lagoon of Venice and a coastal area in the Gulf of Trieste (Northern Adriatic Sea) were maintained at current and predicted level of pH (pH reduction of 0.4 units) for 8 months. Monthly, physiological (respiration rate, ammonia excretion, O:N ratio) endpoints were investigated. Moreover, at the end of the exposure, coelomic fluid, gonads and digestive tract were sampled. The gonadosomatic index (GSI) and a battery of cellular and biochemical biomarkers were assessed. Both pH and time of exposure significantly influenced sea urchin physiological responses, but differences between sites were highlighted, particularly in the first months. Under reduced pH, ammonia excretion increased and O:N decreased in coastal specimens. Animals maintained at pH reduced of ~0.4 units for 8 months showed acclimation capability with higher GSI and reduced oxidative damage compared to the control condition. Moreover, biomarkers results showed strong differences between the two groups of animals. P. lividus from the Lagoon of Venice, which is a more physico-chemical variable environment, showed to acclimate faster to the pH reduction. Lastly, differences between sexes were highlighted with a different modulation of specific biomarkers.

Adaptive Traits in HMA versus LMA sponge holobionts under Ocean Acidification
PRESENTER: Jana Efremova

ABSTRACT. Ocean acidification is affecting our world’s oceans. The steadily increasing absorption of anthropogenic CO2 into the ocean is predicted to decrease seawater pH by 0.5 by year 2100. The impact on marine biochemistry is profound, resulting in a net loss of biodiversity. Benthic organisms must develop adaptive strategies to cope with this stressor. Marine sponges are often forecasted as winner taxa, which will not suffer serious decline in response to increased acidification. Yet, we lack a mechanistic understanding of how these taxa tolerate high acidity and this may not be general to all species. The holobiont concept aims at assessing the sponge host and its functional associated symbiotic microbiome as a metaorganismal unit, when evaluating health, adaptive traits and resilience to environmental challenges. Sponges can therefore be categorized into high and low microbial abundance (HMA, LMA) species, depending on microbial densities in their mesohyl matrix. The HMA status implies more stable and diverse microbial communities, a denser mesohyl and more complex aquiferous systems composed of narrower and longer water channels. LMA sponges rely on simpler aquiferous circuits, which move larger quantities of water, to supply their nutritional needs based on particulate organic matter. The increased water turn-over exposes LMA species to more external stressors, such as changes in seawater chemistry. This may imply divergent strategies to maintain homeostasis and fitness under changing conditions in both sponge groups.

Here we compare the microbial composition of the HMA sponge Chondrosia reniformis and the LMA sponge Spirastrella cunctatrix collected from a natural CO2 vent area with lowered pH (≈ 7,6), and from a control site with normal pH values (≈ 8,1) off Ischia island (Italy). Untargeted metabolomics further reveal the consequences in acclimatizing to ocean acidification for metabolite production.

Impact of ocean acidification on shell morphogenesis in Mytilus galloprovincialis
PRESENTER: Rémi Dumollard

ABSTRACT. One challenge in global change biology is to identify the mechanisms underpinning physiological sensitivities to environmental change. Because coastal carbonate chemistry conditions can fluctuate on various temporal scales, we have studied the impact of variable pH treatment (range 7.4-8.1) on embryonic development and shell morphogenesis of the Mediterranean mussel Mytilus galloprovincialis. Strikingly we have discovered that larval development is impacted by timing of exposure to low pH, revealing sensitivity of two developmental processes: development of the shell field, and transition from the first to the second larval shell. Fluorescent staining revealed developmental delay of the shell field at low pH, and abnormal development. RNA sequencing and in situ RNA hybridization were used to identify processes associated with abnormal development, and DNA sequencing was used to identify which processes may evolve when larvae are exposed to low pH for the full duration of their larval stage. Trochophores exposed to low pH exhibited 43 differentially expressed genes. Thirteen genes, including three unknown genes, were found to be expressed in the shell field. These observations demonstrate that gene expression in the shell field is strongly affected by reduced seawater pH. DNA sequencing revealed that, among known pH sensitive genes, five genes demonstrated changes in allele frequency throughout larval development, indicative of the potential for rapid adaptation. Together, our study demonstrates that ocean acidification affects developmental processes and molecular pathways in Mytilus embryos and that genetic variation in mussel populations may allow this calcifying species to adapt to the changing environment.

17:00-19:00 Session POSTER: APERITIF POSTER
Looking for neuroendocrine components in early development of Mytilus galloprovincialis as possible targets for endocrine disruption
PRESENTER: Beatrice Risso

ABSTRACT. Marine bivalves, living in changing environments like coastal areas, need to cope with a variety of biotic and abiotic stressors. In order to do so, they have evolved sophisticated stress response mechanisms, in which neuroendocrine regulation plays an important role. Various neurotransmitters, hormones, neuropeptides, cytokines have been characterized as signal messengers/effectors regulating different physiological functions such as immune response, energy metabolism, larval development, in response to a vast array of environmental stressors. Bivalve larvae are particularly susceptible to environmental changes, especially during early developmental stages. Different components of neuroendocrine signaling have been shown to play a key role in shell formation in oysters and mussels, and to represent a target for environmental stressors such as ocean acidification and exposure to endocrine disrupting chemicals (EDCs). In this work, expression of components of serotonin and dopamin signaling (receptors, enzymes, transporters) was investigated in early development of Mytilus galloprovincialis (Mg). Target sequences were isolated from Mg genome4. Orthology assessment was performed with respect to human and fruit fly sequences by Maximum Likelihood phylogenetic analysis (MEGAX10.2.6). Gene expression dynamics from 4 to 72 hpf were investigated on the genome guided transcriptome assembly of Mg larvae by stage-in-stage differential expression analysis. Localization of serotonergic and dopaminergic components was evaluated by ISH. Ontogeny of other key components in neurodevelopment was also investigated (other monoamines, enkephalinergic, cholinergic, adrenergic, etc). The results provide first information on the neuroendocrine regulation of functions in early embryo-larval development of the model organism M. galloprovincialis, and will help identifying the most critical steps in neurodevelopment that may represent possible targets for EDCs.

Can Spondylus gaedoropus (Linneaus, 1758) be considered a bio-indicator through its immune system study?
PRESENTER: Federica Arrigo

ABSTRACT. Spondylus gaedoropus (Linnaeus, 1758) is an East Atlantic-Mediterranean sessile bivalve. It preferentially settles in the infralittoral zone, up to 50 m depth. It is also called “spiny oyster”, due to its spinose shell surface having a mymetic function and predator defence. The aim of this study was to understand the ecological role of S. gaederopus through cell description and characterisation to investigate its possible function as a bio-indicator. The animals were collected from theNorthEasternTyrrhenian coast of Sicily (46°19’’3.40”N, 10°48’05.93”E). After 15 days of acclimatation, haemolymph was collected from the adductor muscle using a syringe soaked in 0.4% sodium citrate in a physiological solution. Cells characterizations were assessed using several dyes such as 3% GIEMSA, Neutral Red, Ehrlich triacid stain, Pappemheim stain methods, and it was also detected the lipidic component of membrane using Sudan B Black staining. Enzymatic activity was analyzed by detecting the positivity of haemocytes to acid and alkaline phosphatase, chloro-acetyl acetylase, β-glucuronidase, aryl-sulphatase. Finally, it was also evaluated the capability of cells to phagocytise yeast cells (Saccharomices cerevisiae). The mean number of circulating haemocytes was 3.97± 0.5 cells 10^6 〖mL〗^(-1), and they were able to phagocytise yeast cells. Two cell populations, granular and agranular cells, were detected. In this regard, the presence of granules confirms the presence of hydrolytic enzymes mentioned above. Hyalinocytes are typical cells of mollusks, but in S. gaederopus were not identified. Based on the results obtained, S. gaederopus seems to be an efficient model organism in the field of environmental biomonitoring studies.

Changes in cell viability and volume after chiral pesticide Tebuconazole exposure to Mytilus galloprovincialis (Lamarck, 1819)

ABSTRACT. Tebuconazole (TEB) is a triazole fungicide used in agriculture to treat many fungal diseases worldwide. Due to its extensive use, TEB has now been considered a contaminant of aquatic ecosystems. It is paramount to understand its damage mechanisms to the soil, the aquatic environment, and especially non-target organisms. In particular, marine invertebrates such as Mytilus galloprovincialis, being filter-feeding organisms, are a non-target exposure for tebuconazole. This study aimed to evaluate the effects of subchronic exposure (10-20 days) on the cell viability of M. galloprovincialis and the physiological alterations in the regulatory volume decrease (RVD) of digestive gland cells. The organisms were sampled from Faro Lake, in Messina (Italy), which is commonly used for bivalve breeding. Two different concentrations of tebuconazole (2ng/L and 2µg/L) plus control were chosen for the study. After an acclimatization period, animals were exposed to the pollutant and monitored at 10 and 20days of exposure. Cells from the haemolymph and digestive gland were investigated with the Trypan blue (TB) exclusion method and neutral red (NR) retention test. After 10-d, the cells, while retaining the ability to regulate their volume, varied their swelling and size recovery peaks. The results showed more than 95% cell viability for both sampling times. After 20-d, as supported by other work, the cells that showed the most damage were those exposed to a lower concentration, which lost the ability to swell and recover their cell volume. On the other hand, the cells exposed to the higher concentration reached far greater peaks of swelling than the control, consequently losing their ability to recover. In conclusion, the study showed that while TEB does not affect cell viability in Mytilus galloprovincialis, it negatively intervenes in cell volume regulation, causing damage to the organism.

Could Pinctada imbricata radiata (Leach, 1814) be a model species for ecotoxicological studies?

ABSTRACT. The pearl oyster Pinctada imbricata radiata (Leach, 1814), although native to the Pacific Ocean, was one of the first “Lessepsian migrants” that reached the Mediterranean Sea through the Suez Canal. Due to its extraordinary adaptation, it had the opportunity to spread up also to the western basin. The species can adapt to high ranges of climatic, hydrological, and ecological conditions, which allows it to settle in habitats quite different from the native area, even if characterized by polluted water. It is abundant in the natural reserve oriented “Laguna di Capo Peloro” (Messina), particularly in the canals characterized by high hydrodynamics. Due to the resistance of the species, several assays were carried out on circulating haemocytes to investigate their role in immune responses. The aim of these analyses was to provide a better understanding of the haemocytes population and to propose the species as a model organism in environmental biomonitoring. The following morpho-functional aspects were assessed: cell amount, cell characterization following staining with a variety of dyes, intra- and extra-cellular lipid attendance, cell capability to phagocytize yeast cells (Saccharomyces cerevisiae), and to produce superoxide anion (O2-). Positivity of haemocytes to enzyme activity, such as acid and alkaline phosphatase, aryl-sulphatase, chloro-acetylesterase, and β-glucuronidase was also investigated. Cells proved to be abundant, with a mean value of 8.263 x 106 mm2 ± 0.935 x 106 (SD) and seem to be rather big, in a range between 8-19 μm in size. Two main cell populations were detected: granulocytes and hyalinocytes, which are both phagocytic competent, even if granulocytes appear to be more active in phagocytosis. According to granule content, constitutive enzyme activity was also demonstrated. Based on these preliminary results, we can state that P. imbricata radiata is a promising bioindicator in natural and anthropogenic stressed aquatic environments.

Living under CO2 pressure: investigation of oxidative stress enzymes and metabolic markers in two calcifying mollusks from the CO2 vents of Ischia island (Italy)
PRESENTER: Lorenzo Federico

ABSTRACT. The effects of ocean acidification (OA) are becoming one of the main investigative targets for understanding the anthropogenic impacts on the marine environment. Particularly, we focused on naturally acidified systems, such as the CO2 vents at Ischia, as a natural laboratory to study the long-term effects of OA on calcifying organisms. Our target taxa are the limpets Patella spp., which are long-time residents in the vents, and the mussels Mytilus galloprovincialis, that occur only as juveniles in the vents. Our aim was to understand the potential mechanisms underpinning the acclimatization processes occurring in response to OA in these calcifying taxa. Populations from both taxa were sampled in two sites of the CO2 vent system of Castello Aragonese featured by a natural OA gradient, respectively at mean 7.76 pH and mean 6.90 pH, and compared with a control site, off from the venting area, at pH 8.01. We than investigated for each limpet and mussel populations the activity of electron transport system and the glycogen content as parameters related to metabolic capacity, some antioxidant enzymes and oxidative damage such as lipid peroxidation. Furthermore, untargeted metabolomics analysis was carried out on mussels. We observed a general increase of antioxidant enzymes according to the pH gradient of the vent system in both M. galloprovincialis and Patella spp., with higher level of activities measured in organisms collected in the site at lower pH. Furthermore, a decrease of glycogen content has been observed in M. galloprovincialis along the pH gradient, with lower content detected organisms collected in the site at lower pH, assuming a cost to cope with OA. The metabolomic analysis confirmed an energetic cost related to living under OA.

Mytilus galloprovincialis: a non-target organism as a tool for assessing the potential toxicity of Acetylsalicylic acid (ASA)

ABSTRACT. Pharmaceutical products are largely used by humans, making them commonly found in the environment, especially the aquatic ones. The presence and the increasing concentration represent a risk for non-target organisms and humans too because many of these species enter the human food chain. At the top of the list of medicine and anti-inflammatory drugs, there is acetylsalicylic acid (ASA). One of the main animals present in the human diet is the mussel (Mytilus galloprovincialis Lamarck, 1819) chosen as a model organism for this work. Samples of M. galloprovinciaplis were exposed to two different concentrations of ASA (10 and 100 μg/L) for 10 and 20 days to evaluate potential variation in three different organs: the digestive gland, haemocytes, and gills. We analysed: • Viability tests by Trypan Blue (TB) and Neutral Red (NR) of haemocytes and cells of digestive glands. • Regulation of volume decrease in digestive gland cells • Histological condition index of gills and digestive gland. The results obtained showed that ASA alters the physiological mechanism of volume regulation in the digestive gland and that several inflammations in this organ and the gills are present. The data acquired to confirm the potential toxicity of ASA in the aquatic environment, highlighting the importance to improve the analysis of the effects of the pharmaceutical products and non-target organisms.

Innovative biomonitoring in port ecosystem: lessons and perspectives from the QUAMPO project

ABSTRACT. The rapid modification of coastal landscapes is on course to reach a critical point and port areas symbolises the outmost of such landscape alteration. The growing trend in recreational boats whose market grow continuously in the coming years also leads to the anthropization of coastal habitats through the development of ports. By their coastal settlement, ports replace important natural habitats for biodiversity but can still bear surrogate artificial habitats from nursery to refuge areas. Unsurprisingly, biodiversity in port ecosystems faces many anthropic pressures from port activities and has been under-studied by lack of interest of local stakeholders and a difficult environment. Difficulties in implementing integrated environmental management, accounting for the great complexity of the marine socio-ecological systems, have been part of the issue. Difficulties in engaging and convincing private stakeholders and a number of economic sectors involved in these processes in ports for more sustainable practices have also contributed. This reticence is traditionally more pronounced in the port sector, despite their important role in the transformation of coastal areas. However, the maritime industry and port areas have been under scrutiny regarding their environmental impacts and the development of environmental certifications is providing incentives to engage port managers in the implement of actions to fulfil these environmental certifications and European Directive requirements. This paper seeks to assess the constraints posed on different port dynamics for managing and ultimately mitigating the port environmental impacts. We present the outcomes and lessons learned from the QUAMPO project and highlight the opportunities to move towards port biodiversity restoration in Corsica using innovative biomarker monitoring.

Nanoplastics impact on early development of Ciona robusta embryos and juveniles at environmentally realistic concentrations
PRESENTER: Emma Ferrari

ABSTRACT. Plastic pollution is recognized as a global threat for natural ecosystems and recent findings documented the occurrence of the smallest fraction named as nanoplastic (<1μm) in marine waters, including marine coastal areas of the Mediterranean Sea. The smallest size rebounds to their safety associated with remarkable biological, chemical and physical reactivity that allow them to interact with the cellular machinery by crossing biological barriers and causing damage to living beings. Therefore, the understanding of bio-nano interactions, occurring with the most sensitive early life stages of marine species, is essential to predict nanoplastic impact and make predictions on cascade events at community level. The ascidians, as chordate marine model having chorionated embryos, could be very useful to assess the impact of nanoplastics and predict also scenarios for higher chordates as fish. Here we assess the role of un-charged polystyrene nanoparticles (PS, 20 nm) as proxy for nanoplastics on Ciona robusta embryos/larvae development under realistic environmental concentrations and scenarios (10-50 µg/L) by looking at several endpoints as: lethality, fertilization rate, hatching, growth and larvae viability and motility. No effects on C. robusta embryos development were observed and besides the hatching success, no phenotypic alterations up to juvenile stage (4 dfp) nor structural modification. However, a mechanical slowdown in the swimming behaviour of the larvae was observed likewise, at the juvenile stage, an accumulation of PS NPs in the organisms' inhalant siphons, which could slowdown filtration and affect feeding. Our preliminary findings suggest bio-nano-interactions occurring between nanoplastics at environmentally realistic exposure concentrations and ascidians embryos later after hatching thus suggesting that prolonged exposure scenarios could unravel potential detrimental impact on early life stages of marine life.

PRESENTER: Tatiana Rusconi

ABSTRACT. The increasing anthropic pressure into the marine environment has led to a progressive degradation of ecosystems health. From various sources and origins, nanoscale materials end up into the marine environment and pose a treat for marine biodiversity. Nanocellulose is been increasingly used in many industrial and environmental applications, therefore it is expected to be significantly released in water bodies. Recent findings on the presence of micro and nanofibers (NF) in Mediterranean species, mostly of cellulose-based composition, raise concern on their potential ecotoxicity despite its natural polymer composition. The environmental safety of cellulose nanofibers (CNF) and TEMPO-oxidized CNF (TOCNF) was here investigated in terms of behavior and ecotoxicity on the marine mussel Mytilus galloprovincialis. Dispersion and morphology of CNF and TOCNF were evaluated in artificial and natural sea water (A/NSW). The uptake of CNF and TOCNF was tested in vivo (96h) with mussels exposed with labeled CNF/TOCNF (1 µg/L, 1 mg/L). A battery of biomarkers in mussel’ digestive gland, gills and hemolymph as indicators of cytotoxicity, neurotoxicity, oxidative stress and phase 0 of biotransformation were investigated upon an in vivo acute exposure (48h) to CNF and TOCNF (1 µg/L, 1 mg/L). TOCNFs in NSW undergo higher fragmentation upon sonication compared to CNF. Uptake of either CFN and TOCNF was confirmed in hemolymph and gills, with the latter prevalent in hemolymph and mantle edges. Biomarkers indicative of oxidative stress and detoxification were not triggered, however, a bio-interaction of CNF and TOCNF with circulating hemocytes and gill cells was found, more evident for TOCNF than CNF. Inhibition of ATP-binding cassette efflux pump in gills and destabilized lysosomes and reduced acetylcholinesterase activity in hemocytes were observed. CNF and TOCNF were not toxic for marine mussels but their uptake and bio-interactions suggest further investigation for risk assessment.

Integrated biological and chemical approach to evaluate water quality against contaminants of emerging concern
PRESENTER: Paola Valbonesi

ABSTRACT. Chemical assessments of Contaminants of Emerging Concern (CECs) are requested by the recent updates of Directives in the field of water policy and quality of water intended for human consumption (EU 2020 and EU 2022, respectively), where prioritization of compounds is achieved through the Watch list mechanism. However, the adverse biological effects from mixtures of substances present in the water may not be predictable on the basis of chemical analyses alone, suggesting the need for coupling effect-based methods to chemical assessments. In the present study chemical analyses of a selection of CECs (i.e. 5 pharmaceuticals, 8 endocrine disruptors) have been carried out by HPLC-MS/MS on water samples collected over a five-year period at entrance (untreated) and exit (treated) of three treatment plants serving the Romagna region (Italy) managed by Romagna Acque Società delle Fonti. Complementary biological analyses were performed to ascertain treated water estrogenic and/or genotoxic potential, by E-screen and Micronuclei assays, respectively. Bisphenol A (BPA), nonylphenol (NP) and perfluorooctanoic acid (PFOA) were the most frequently detected endocrine disruptors, found in treated water at: BPA <LOQ - 6.27 ng/L, NP 7.90 - 104.87 ng/L and PFOA <LOQ - 12.66, ng/L. Among pharmaceuticals, caffeine (CFF) and carbamazepine (CBZ) were the most often detected contaminants in treated water (CFF <LOQ - 92.80 ng/L and CBZ <LOQ - 1.19 ng/L). Overall, treatments were efficacious in reducing BPA, CFF and CBZ, while sometimes were ineffective for NP and PFOA. No estrogenic or genotoxic activities were observed in treated water, except for one sample showing a weak estrogenic activity, quantified as 9.06 pg/L EEQ. In conclusion, although the water analyzed was of high quality, this study confirms that a battery of effect-based tests provides advantages in the monitoring of water systems, by detecting effects of mixtures of pollutants not disclosed by chemical analysis.

Mytilus early larval development : from the embryotoxicity assay to AOP of contaminants of emerging concern
PRESENTER: Teresa Balbi

ABSTRACT. Bivalve early developmental stages are most sensitive to different environmental stressors. The standardized 48h embryotoxicity assay, based on microscopical determination of the percentage of normal D-larvae developed at 48 hpf, can be utilized to test chemicals or water samples in common bivalve species (mussels, oysters, clams)1. The assay was adapted to 96-multiwell plates2 for high throughput screening of Contaminants of Emerging Concern (CEC), tested in a wide concentration range (ng-µg/L) in Mytilus galloprovincialis. Here we summarize the results obtained so far with Endocrine disruptors (estrogenic compounds, brominated and perfluorinated compounds, Tributyltin, Triclosan); Pharmaceuticals (non steroid anti-inflammatory drugs-NSAID; antidepressants including Selective Serotonin Reuptake Inhibitors-SSRIs); Nanomaterials: metal-based, nano-oxides, surface modfiied nanopolystyrenes. Although the results can provide significant amounts of data potentially useful for regulatory purposes, the endpoint of this simple ecotoxicological assay does not allow for the identification of more subtle changes induced by contaminants that may affect subsequent development (i.e. alterations in shell biogenesis and neurodevelopment). On the other hand, embryotoxicity data can represent the basis for investigating the mechanisms of action of different CEC at environmental concentrations, from molecular to organism level, and contribute drawing Adverse outcome pathways (AOPs). Assessment of ecosystem health with respect to CEC is of particular concern in the marine environment: in this respect, data on the effects on early life stages are important to establish the sensitivity of marine invertebrate species.

Triclosan as a potential developmental disruptor in Mytilus early larvae
PRESENTER: Teresa Balbi

ABSTRACT. Triclosan (2,4,4’-trichloro-2’-hydroxydiphenyl ether, TCS) is an antimicrobial agent present in a variety of personal, household and healthcare products. Due to its incomplete removal by the conventional wastewater treatment plants is it continuously discharged in water worldwide, and its concentration in coastal areas due to river inputs is expected to increase in the next decades. Moreover, the use of TCS-containing products during the COVID-19 pandemic is likely to have increased its content in aquatic ecosystems. TCS is considered as a potential endocrine disruptor in multiple species, and it is included in the list of substances undergoing an ED assessment under REACH or the Biocidal Products Regulation. Although ecotoxicological data indicate a PNEC of 26.2 ng/L in aquatic organisms, TCS has been reported to be accumulated and induce a variety of effects in marine organisms, in particular bivalves. In this work, the effects of TCS on early larval development of Mytilus galloprovincialis were investigated. The results indicate that TCS can affect Mytilus normal development of 48 h larvae from 10 ng/L. TCS affected shell formation, as shown by SEM images and expression of genes involved in shell biogenesis. Moreover, key genes involved in neurodevelopment were evaluated. The results demonstrate that Mytilus early development represents a significant target for environmental concentrations of TCS. Moreover, the data obtained represent the basis for investigating the molecular mechanisms underlying the developmental effects of endocrine disrupting chemicals in marine invertebrates.

Antiproliferative effects of methanolic extracts of two marine sponges on pancreatic cancer cell line
PRESENTER: Maria Costantini

ABSTRACT. Although the Mediterranean Sea covers approximately a 0.7% of the world’s ocean area, it represents a major reservoir of marine and coastal biodiversity. Among marine organisms, sponges (Porifera) are a key component of the deep-sea benthos, widely recognized as the dominant taxon in terms of species richness, spatial coverage, and biomass. Sponges represent the most studied marine organisms as sources of bioactive compounds. In fact, previous studies reported the bioactivity of different marine sponge extracts in several diseases and some extracts have been used to produce commercial anticancer drugs. In the present study we tested the biotechnological potential of two marine sponges, Oceanapia cf. perforata (Sarà, 1960) from Faro Lake in Sicily and Agelas oroides (Schmidt, 1864) from Punta San Pancrazio in the Gulf of Naples. In our previous studies these two sponges were characterized by morphological analysis and amplification of several conserved molecular markers. Here we identified active fractions from both sponges able to block the proliferation of the human pancreatic cancer cell line (PaCa-2). Further bioassay-guided fractionation and chemical characterization will be performed, aiming to identify natural products from both sponges responsible of this activity.

Bioaccumulation and synergistic effects of chronic pesticide pollution on the model species Mytilus edulis: A model for catchment-based risk mapping.
PRESENTER: Graihagh Guille

ABSTRACT. The human global population is predicted to reach 9.2 billion by 2050, requiring a 70% increase in agricultural output. Synthetic chemical pesticide use has vastly increased food crop yield since the 1940s, however they are now contaminating surface water bodies, playing a significant role in the high extinction rate of fresh-water species. The 2021/22 report on water quality in rivers within England, states that only 14% of rivers within England have “good ecological status” while 0% have a good chemical status. Current regulations limits the individual and sum of pesticide concentrations in surface water bodies, however, they do not consider synergies and antagonisms. This study, supported by South-East Water, will employ passive sampling and biomonitoring, in combination with environmentally relevant laboratory experiments, to evaluate the risk of pesticide mixtures, present in a surface water body catchment, to aquatic organisms. Bi-monthly grab sampling data have been analysed to identify key dates, locations, and target analytes for passive sampling. These data were highly correlated with the agricultural seasonal trends and application practices, peaks were detected mainly between April and August. Passive samplers (Chemcatchers) have been deployed alongside caged mussels (Mytilus edulis). The study will provide accurate information on the pesticide’ levels in the water and their bioavailability to aquatic organisms. Subsequent laboratory exposures of mussels at different life stages will use relevant compounds and concentrations in mixtures. Biomarkers of oxidative stress/endocrine disruption will be used to calculate deviations from additive toxicity. Models for risk mapping will be adapted to combine data on the location, seasonality and concentration of pesticide mixtures and their associated harm to the model species M. edulis. The impact of environmentally relevant concentrations of pesticides and their synergies on freshwater biota will be discussed.

Ecotoxicological impact of plastic leachates on Mytilus edulis haemocytes

ABSTRACT. Micro(nano)plastics contain complex mixtures of additive chemicals that can be potentially hazardous. These compounds are not covalently bound to the plastic polymers and during weathering processes they can leach out to the environment. Additives include flame retardants, metals, bisphenols, and organic chemicals, some of which belonging to the list of known estrogenic compounds and emerging contaminants. Thus, there are increasing concerns regarding the potential toxicity of these chemicals on marine organisms. Moreover, detailed knowledge on how additive-containing leachates from plastic can possibly harm marine life is still lacking. Bivalves’ haemocytes are directly exposed to environmental stressors and are involved in both humoral and cellular mechanisms, playing a key role in immunological homeostasis of invertebrates. In this framework, the present study focuses on comparing the ecotoxicological impact of leachates derived from commonly used plastic products to Mytilus edulis haemocytes by applying a reproducible flow cytometry methodology that mimics an in vivo state. Haemocytes were exposed for 24 h to leachate dilutions originating from balloons, gloves and shoes soles. Biological endpoints assessed encompass mortality, cell viability, reactive oxygen species (ROS) formation, cellular and mitochondrial membrane potential, lipid peroxidation (LPO), neutral lipid content, esterase activity, lysosome presence and DNA damage. Provisional data seem to indicate significant toxicity of leachates in haemocytes, even at low concentrations. This study provides new knowledge on the toxicity pathways of plastic leachates in the aquatic environment and highlights the importance of comprehensively understanding the potential environmental risk of plastic pollution.

Acknowledgement – This work was supported by the Stipendiatstilling program (grant number: 321173) and the project MicroLEACH (grant number: 295174), funded by the Norwegian Research Council.

Insight into the role of ovothiol biosynthesis in response to environmental pollutants in cnidarian species

ABSTRACT. Ovothiols are histidine derived thiols, endowed with peculiar redox properties, originally found in marine invertebrates, protists and bacteria. Previous studies demostrated that ovothiols finely regulate the intracellular homeostasis of sea urchin larvae in response to chemical pollutants dispersed in seawater. The enzymes involved in ovothiols biosynthesis, the 5-histidylcysteine sulfoxide synthase OvoA and the pyridoxal phosphate-dependent lyase, have been recently characterized. Although this biosynthetic pathway is conserved in metazoa, ovoA gene loss events occured in ecdysozoa and in vertebrata, and two cases of ovoA secondary acquisition through horizontal gene transfer (HGT) were discovered in bdelloidea rotifera and hydrozoa. All these events contributed to the complex evolutionary history of ovothiols biosynthesis, whose diversification, distribution and ecological roles remain still unveiled. This study aims to gain insight into the evolution of ovothiols biosynthesis in cnidarians and in the involvement of such molecules in coping with enviromental change conditions. Thanks to the availability of public genomic and trascriptomic data, we identified cnidarian species endowed with this secondary biosynthetic pathway, reporting another case of HGT in the hydrozoan Clytia hemisphaerica. Through comparative trascriptomic analyses, we showed different ovoA expression levels during embryo and larval development of different cnidarian species, and in response to enviromental polluttants, suggesting a functional role of ovothiols in such a delicate phase of life cycle and their involvement in the protection of marine organisms against environmental stressors. Finally, we traced the occurence of 5-thiohistidine derivatives in anthozoan and medusozoan animals through high performance-liquid cromotography analyses. Our work provides new knowledge to unravel the origin, diversification, and ecological role of ovothiols biosynthesis.

Risk assessment in the Anthropochemical era: A receptor-based tool for the identification of Endocrine Disrupting Chemicals

ABSTRACT. In the Anthropocene, wildlife is critically impaired by environmental chemical build-up and off-target effects. Currently, ecotoxicological risk assessment (RA) of pollutants relies significantly on the extrapolation of physiological responses across taxa, to predict how molecular pathways, individuals and ecosystems are affected, and ultimately to improve regulatory efforts, and management measures. Nuclear receptor (NR) based in vitro assays are recognized as major RA tools, being already included in several frameworks, and soon to be recommended by the EU Water Framework Directive. Yet, the representativeness of such responses across invertebrates is still challenging, limiting the accurate inference of the taxonomic scope of adverse outcome mechanisms. A prime example, within the NR superfamily, is the ligand-induced ecdysone receptor (EcR): A transcription factor that triggers molting as a response to hormonal signals. For long, it was wrongfully considered arthropod specific and thus, greatly exploited for the development of insecticides, labelled safe for non-target organisms. However, the unveiling of a broader presence of EcR orthologues across invertebrate lineages marks such compounds as potential endocrine disrupting chemicals (EDCs), known to interfere with endogenous hormones or mimic them, altering signaling pathways and producing adverse effects to non-target organisms. In this work, we made use of luciferase-based transactivation assays to functionally characterize the EcR of two sentinel amphipod species. Apart from highlighting differences in EcR’s transcriptional activity between both species, when exposed to selected compounds, our results also identified possible off-target EDCs, including two diacylhydrazine insecticides. This strengthens the need for proper assessment of NR function and modulation in representative species, not only to fill out knowledge gaps, but also to develop phylogenetically relevant RA tools to protect ecosystem webs.

Erythrocytic dynamics and kidney oxidative condition show an adaptive response of Scophthalmus maximus to personal care products
PRESENTER: Cláudia Mieiro

ABSTRACT. Personal care products (PCPs), including chemicals such as UV filters and fragrances, can reach water systems and are persistent, bioactive, and harmful to aquatic biota. Despite that, research has mainly focused on freshwater scenarios and a reduced range of effects. So, our goal was to evaluate the effects of an inorganic UV filter (titanium dioxide nanoparticles), an organic UV filter (4-methylbenzylidene camphor - 4-MBC) and a synthetic musk fragrance (galaxolide - HHCB) on the hematological dynamics and kidney oxidative stress profile of the marine flatfish Scophthalmus maximus. PCPs were intraperitoneally (IP) injected, individually and as binary and ternary mixtures, in concentrations based on those found in the environment. Red blood cell count (RBC) and erythrocytic maturity index (EMI), as well as the antioxidant profile (CAT, GPX, SOD and GR) and lipid peroxidation (LPO) in the kidney, were evaluated 3- and 7-days post-injection. On day 3, RBC increased in the mixture of 4-MBC + HHCB relatively to control and to their isolated exposures, while EMI displayed a decrease of class 5 in the same treatments and the ternary mixture. No alteration of the kidney antioxidant profile occurred, but LPO decreased in all treatments. These results on day 3 reflect adaptive responses, depicted on the adjustment of hematological dynamics and increased RBC, as a fish strategy to cope with a higher demand for oxygen after short-term exposure to contaminants. The depletion of LPO in the kidney on day 3, as well as the absence of effects in blood and kidney on day 7, strengthened the interpretation of the fish's ability to resist the current PCPs challenge. Overall, the injection of low concentrations of these PCPs triggered adaptive pathways, while no increment of toxicity emerged amongst the mixtures and the isolated chemicals, pointing to no relevant ecological effects.

Neurotransmitter and endocrine systems in marine invertebrates: targets for medetomidine.

ABSTRACT. Neurotransmitter systems are major elements in the transmission of information within the central nervous system and for its interaction with target organs to regulate physiological functions. These systems are highly conserved in the animal kingdom. Therefore, neurotoxic compounds could interact with similar neurological and neuroendocrine pathways in vertebrates as invertebrates. In invertebrates, such as mollusks and crustaceans, monoaminergic neurotransmitters (serotonin, dopamine, norepinephrine, octopamine) modulate the levels of neuropeptide hormones. A perturbation of these systems by emerging pollutants may, therefore, lead to endocrine disruption. Both, the modulation of neurotransmission and of hormonal control may affect behaviour. In order to shed light on neuro-endocrine perturbation in a mollusk, the locomotor activity and camouflage behaviours (colour change and sand digging) were studied in the cuttlefish Sepia officinalis following exposure to medetomidine, a veterinary sedative and an antifouling agent.

Impairment of reproductive structures in the field lizard Podarcis siculus exposed to glyphosate.
PRESENTER: Teresa Chianese

ABSTRACT. Glyphosate (GLY) is a widespread herbicide in agricultural practice applied in formulations containing adjuvants/surfactants; evidence of adversely effects on non-target organisms is accumulating. In this study we exposed the lizard Podarcis siculus to oral administration of pure GLY at two doses (0.05 and 0.5 ng/g body weight every other day for 3 weeks) to evaluate its toxicity on the reproductive functions of this animal, which lives in cultivated areas and therefore potentially exposed. We used pure GLY to attribute our results to the active ingredient. Morphological and molecular analysis demonstrated dose-dependent GLY toxicity. Histological analysis revealed alterations of the seminiferous tubules, characterized by an epithelium with many empty spaces and germ cells forming rosette aggregates, and a lumen occupied by few spermatozoa and degenerated cells; the interstitial spaces showed excessive deposition of connective tissue. Molecular investigations demonstrated that GLY did not affect steroidogenesis but altered the gap junctions of Sertoli cells and changed the expression and localization of estrogen receptors. The ovary also showed significant GLY-induced damage. Theca had thickened connective tissue and a reduction of the cellular component; the follicular epithelium was irregular in thickness and many apoptotic cells were present. Zona pellucida was irregular, with an altered content of glycans. Changes in the content and distribution of glycans were also found in cells surrounding oocytes, whose cytoplasm was denser and rich in filamentous aggregates. We can state that the effects of GLY must be considered biologically relevant and could endanger the reproductive capacity of lizards and possibly other vertebrates. Further studies are needed to identify the molecular pathways underlying the effects of GLY and to investigate the impact of co-formulants, as we cannot exclude the possibility of synergistic interactions between GLY and other chemicals.

Benzodiazepine inhibits feeding behaviour while inducing hyperactivity and altering pedal gland characteristics in the freshwater gastropod Planorbarius corneus

ABSTRACT. Benzodiazepines are among the most diffuse emergent pollutants, and their presence in aquatic environments all over Europe has reached dangerous levels. Benzodiazepine's adverse impacts on freshwater species have been documented. Here, we report preliminary data on the effects of benzodiazepine on the freshwater gastropod Planorbarius corneus, a diffuse species in the central European floodplain and wetland, although constantly declining. We tested the effects of 4 days of exposure of adult specimens (shell size 15-20mm) to 10 µg/L of a commercial benzodiazepine product (delorazepam, oral drops, 1 mg/ml). We evaluated the effects on animal behaviour in thigmotactic and feeding performance tests. Compared with controls, treatment with benzodiazepine induced no qualitative effect on thigmotaxis but inhibited feeding activity and strongly increased locomotion velocity. Since locomotion in snails depends on the combined action of muscles and mucus, histological analysis of the foot was performed. Results reveal no apparent benzodiazepine-dependent alteration in muscles or pedal glands structure but suggest that treatment increases glands secretions and alters mucus characteristics. Overall, the results prove that benzodiazepine induces in adult Planorbarius corneus hyperactivity associated with altered pedal morpho-functional properties likely related to the higher locomotor speed. More importantly, the hyperactive response was related to the inhibition of the feeding behaviour that could draw to fatal consequences if prolonged.

Interaction of natural organic matter with Bisphenol-A in water phase: preliminary observations and its implication on toxicity

ABSTRACT. This study presents an experimental study aimed at investigating the mechanism of Bisphenol-A (BPA) interaction with Natural Organic Matter (NOM), the mobility of contaminants in the aquatic environment, the evaluation of bioavailability, and cytotoxicity. The high-performance size exclusion chromatography shows NOM aggregates itself in water into non-spherical three-dimensional structures with sizes ranging within the nanometric scale. High-performance liquid chromatography and fluorescence analysis reveal that the NOM strongly interacts with BPA. Such moieties interact stably with the contaminant probably due to the establishment of dispersive forces such as π-stacking which sequestrate BPA into the inner hydrophobic core of the organic matter three-dimensional structure, surrounded by hydrophilic moiety formed by progressive aggregation of amphiphilic and polar molecules, mostly deprotonated carboxylic groups. With regards to the NOM/BPA interaction, the invertebrate Mytilus galloprovincialis was selected for the NOM/BPA effect in eliciting noxious effects on marine biota. The observation of the gonadal sections of male Mytilus galloprovincialis after 96 hours with NOM/BPA complexation and BPA alone have distinct differences. With NOM/BPA addition, samples are evident full spermatic cysts with germinal cells in different stages of differentiation. However, with BPA addition, empty spermatic cysts with residues of germinal cells and the presence of immune cells are evident, which evidenced a NOM protective effect that is able to counteract BPA toxicity on the male gonadal organization of adult Mytilus specimens. The biological relevance of NOM/BPA interaction was investigated in human intestinal cancer cells SW480 and HCT116 cell lines. The results indicate that NOM blocks the effect of BPA whereas BPA itself stimulated cell growth. It can be assumed that NOM by complexing with BPA reduces its bioavailability, exerting a protective role against a toxic compound.


ABSTRACT. Marine protected areas (MPAs) are sea regions where legal restrictions on human activity are applied for conservation purposes, protecting marine resources from human activities. However, they do not guarantee protection from environmental anthropogenic pollutants. The assessment of water quality and pollution levels, and the identification of suitable bioindicator species as sentinel organisms could improve the effectiveness of MPA management. Biomarkers of oxidative stress, i.e., unbalance between reactive oxygen species (ROS) production and antioxidant defense systems, link to altered animals’ physiology with potentially detrimental consequences on other species in the community. Thus, they are used in pollution monitoring, enabling the establishment of cause-and-effect relationships. In this study, we propose the black sea cucumber Holothuria forskali, a widely distributed species in the Mediterranean Sea, as a key species to compare three areas at Punta Campanella MPA subject to decreasing degrees of protection: Ieranto (B* zone, *with additional protection on fishery periods), Punta Campanella (B zone), and Puolo (C zone). We evaluated ROS production, oxidative damage to lipids and proteins, antioxidant enzymes’ activity, and susceptibility to oxidants of the respiratory tree. We also measured heavy metals (Cd, Pb, Cu, Zn) levels in seawater samples collected in the holothurians’ environment. We found the highest levels of ROS production, oxidative damage, and susceptibility to oxidants, not balanced by the antioxidant enzymes activities, in the organisms collected from the C zone, which shows the highest Pb and Cu concentrations. The B* and B areas showed minimal and intermediate redox state alterations, respectively. High Pb concentration was also found in the B area. In conclusion, H. forskali is a suitable sentinel organism to monitor the effectiveness of MPA management, and redox state alterations could allow making decisions to improve the management.

Adaptation of zebrafish gills after aluminium exposure

ABSTRACT. Aluminum is considered a dangerous pollutant, especially for aquatic organisms, since the phenomenon of acid rain increases its availability in water bodies. This study aims to obtain preliminary data on the effects of aluminum exposure on the fish gills, the first organ interacting with the metal in water bodies. Zebrafish, a widely used species in ecotoxicological studies, has been chosen as an experimental model. Adult specimens were exposed to 11 mg/L for 10, 15, and 20 days. We evaluated the in vivo oxygen consumption and tail beats, as well as gill's homogenate COX activity, ROS content, relative ability to scavenge ABTS, antioxidant enzyme activity, oxidative damage to lipids, and in vitro susceptibility to oxidative stress was evaluated. The results indicate that aluminum alters both animals' wellness and oxidative state, disrupting redox homeostasis and impairing oxygen consumption and spontaneous activity. However, after 20 days of exposure, the antioxidant system efficiency increases, suggesting an adaptive mechanism that makes the animal less susceptible to aluminum-induced oxidative stress. Furthermore, after 20 days of exposure, oxygen consumption is reduced, while the tail beats increase, suggesting the onset of anaerobic metabolism as an adaptive mechanism. Overall, the results provide an essential starting point for assessing the toxicity induced by aluminum on the gills of aquatic organisms, the effects of which are still poorly understood.

Metabolic changes in gills and liver of the white seabream (Diplodus sargus) induced by dietary methylmercury exposure

ABSTRACT. Mercury (Hg) is recognized as a dangerous trace element since its persistence in aquatic habitats and high toxicity to organisms have raised environmental and human health concerns. Its organic and highly toxic form, methylmercury (MeHg), easily crosses biological membranes and readily accumulates in tissues of biota. Despite MeHg toxicity has been highly explored in aquatic organisms, understanding the mechanisms of dietary MeHg toxicity in fish remains a challenge. Therefore, a time-course experiment was herein conducted with juvenile white seabreams (Diplodus sargus) exposed to realistic levels of MeHg in feed (MeHg 8.7 μg/g, dry weight), including exposure (7 and 14 days) and post-exposure (28 days) periods. Gills and liver were chosen as target organs because of their distinct functions. Total Hg levels in both organs increased linearly with time during the exposure period and decreased significantly in the 28-day post-exposure. Besides that, liver exhibited a higher level of Hg than gills, particularly in the exposure period. The high-throughput Nuclear Magnetic Resonance (NMR)-based metabolomics revealed multiple and often differential metabolic disorders between fish organs. Specifically, fish liver exhibited an altered protein metabolism with a drop in the level of amino acids, likely due to the activation of defensive strategies. The post-exposure period allowed a partial recovery, even if with occurrence of oxidative stress and changes in energy metabolism. Conversely, dietary MeHg exposure provoked the occurrence of protein catabolism in fish gills, as well as disturbances in cholinergic neurotransmission and changes in osmoregulation, lipid and energy metabolism. Overall, organ-specific cytotoxicity mechanisms of dietary MeHg exposure were discerned, pointing out the vulnerability of fish health to this Hg form at highly impacted ecosystems and the need of increase surveillance in this direction

Sediment plumes impact benthic organisms at multiple levels: a case study using Mytilus galloprovincialis under hyperbaric conditions
PRESENTER: Marlene Pinheiro

ABSTRACT. Land-based deposits are diminishing and driving the growing interest in deep-sea mining to obtain minerals and rare earth elements. Deep-sea environments, despite largely unknown and remote, are exposed to anthropogenic stressors and will be affected by mining operations. Sediment plumes are largely considered as one of the most immediate and widespread consequences of deep-sea mining. To study the potential effects of sediment plumes associated with deep-sea mining using the model species Mytilus galloprovincialis, we studied the effects of different sediment size classes (63-125; 125-250; 250-500um), in a mixture (60, 30 and 10% respectively) with increasing concentrations (1, 2 and 4g/L) at increasing pressures (1, 4 and 50 bar) in a hyperbaric chamber for 96h. We evaluated the filtration rate (FR) and oxidative stress biomarkers (catalase, lipid peroxidation, glutathione S-transferase and superoxide dismutase) in juvenile mussels. As significant changes were found in all tested biomarkers, and the FR decreased for all concentrations tested, we then assessed the effects of the sediment size classes separately. We exposed juvenile mussels to the different classes individually at 1 g/L and 4 Bar. Functional, biochemical, and molecular endpoints were analyzed. The FR decreased significantly for all treatments with a more pronounced effect in the smaller size classes. Significant changes were also found for all biomarkers tested and gene expression was downregulated for actin and catalase. These results indicate that small particles cause adverse effects and since they have high distribution and suspension potentials, particular attention should be given to their release and resuspension, in the water column of the seafloor, as a by-product of mining practices. These findings will provide additional data to create guidelines and protocols to mitigate the negative effects of deep-sea mining in delicate habitats.

Seasonal changes in immune biomarkers in Mytilus galloprovincialis farmed at the Gulf of La Spezia, Ligurian sea
PRESENTER: Martina Leonessi

ABSTRACT. Marine mussels (Mytilus spp.) are used worldwide for biomonitoring with a multi-biomarker approach. Aquaculture bivalves, have been shown to be targeted by a large range of environmental stressors. However, to fully understand biomarker responses, knowledge is needed on their natural seasonal variability, depending by both biotic and abiotic factors. This also applies to biomarkers related to immunity, a key physiological process involved in maintaining the organism health status. In this work, the seasonal variability of several biomarkers from subcellular to whole organism level, was studied over 1 year (February to November 2020) in adult mussels Mytilus galloprovincialis cultivated in the Gulf of La Spezia (Ligurian Sea, Italy). A set of immune parameters was evaluated on hemocytes and hemolymph: lysosomal membrane stability (LMS) as a biomarker of general stress, phagocytic activity, extracellular reactive oxygen species (ROS) and lysozyme activity. Changes in hemolymph microbiota composition were evaluated by 16S rRNA gene amplification and sequencing. Data related to general health condition, determined as mussel survival time in air (stress on stress SoS response), and the gonads maturation stage were also collected. The results show a seasonal variability of immune parameters related to both environmental (variation of the water temperature) and biological (gametogenic cycle) factors. These results demonstrate that mussels can be more susceptible to additional stressful conditions during the spawning season, confirming previous data obtained in mussels from the same local aquaculture farm1. Moreover, the result provides new information on seasonal changes in hemolymph-associated microbiota, and their possible relationship with immune defenses. The obtained information is important for the support of local bivalve aquaculture and its productivity.

Transgenerational effects of ocean warming and deoxygenation on Gammarus locusta
PRESENTER: Beatriz Pereira

ABSTRACT. Elevated temperatures and reduced oxygen availability have been shown to have marked adverse effects on marine biota. Yet, the ability of organisms to rapidly adapt to these stressors has been poorly understood. This study aimed to assess the transgenerational effects of end-of-the-century predicted levels of ocean warming (+ 3ºC) and deoxygenation (90% air sat. O2) on the oxidative stress and hormonal profiles of keystone crustacean, Gammarus locusta. To this end, three generations of gammarids were exposed to present-day and future climate change scenarios, following a full-factorial design. Subsequently, animals were sampled, and HSP, SOD, MDA and ubiquitin were quantified, as well as hormones (GABA, dopamine, acetylcholinesterase and serotonin) and DNA damage. For the first time, these results will untangle the transgenerational effects of ocean deoxygenation and its factorial exposure to ocean warming on an intertidal amphipod.

Microplastics and copper induce apoptosis, alter neurocircuits, and cause behavioral changes in zebrafish (Danio rerio) brain
PRESENTER: Dércia Santos

ABSTRACT. The knowledge regarding the neurological and behavioral toxic effects associated with microplastics (MPs) and heavy metals exposure is still scarce. The present study aimed to evaluate the potential chronic (30 days) toxic effects of MPs (2 mg/L) and copper (Cu, 25 µg/L), alone or combined, in the zebrafish (Danio rerio) brain antioxidant system, cell proliferation/death, cholinergic-, serotonergic- and dopaminergic pathways and, consequently, in locomotor, anxiety, and social behaviors. Our findings showed that MPs and Cu exposure modulated the antioxidant system of zebrafish brain, with superoxide dismutase (SOD) and glutathione reductase (GR) having higher activity in the Cu25+MPs group, but glutathione peroxidase (GPx) being inhibited in MPs, Cu25 and Cu25+MPs. Moreover, an increase in acetylcholinesterase (AChE) activity was observed in all exposed groups. When considering neurogenesis genes, a downregulation of proliferating cell nuclear antigen (pcna) was noticed in zebrafish exposed to the mixture treatment, while for dopaminergic system-related genes (th and slc6a3) an upregulation was observed in MPs, Cu25 and Cu25+MPs groups. An increase in apoptosis-related genes expression (casp8, casp9 and casp3) was observed in the MPs exposed group. Changes in zebrafish behavior, particularly in mean speed, total distance moved, inactivity in the aquaria, and social/shoaling behavior was also observed in the MPs and Cu exposed groups. The IBR index applied to biochemical and genetic biomarkers results showed that the treatments with plastic particles presented the highest index values (2.033 and 2.097 for MPs and Cu25+MPs, respectively), thus reflecting higher stress. Overall, our results highlight the multiplicity of toxic effects of MPs, alone or combined with Cu, in zebrafish brain, namely apoptosis and alterations in adult neurogenesis, neurocircuits and, consequently, behavior.

Effects of textile polyester microfibers on Mytilus galloprovincialis
PRESENTER: Manon Auguste

ABSTRACT. Microplastics vary in material, size and shape, that in consequence will influence the type of interaction, uptake and effect on biota. Textile microfibers (MF) have become a global concern during the last decade since they represent the major primary source of synthetic MF found in the environment. Marine bivalves, due to their suspension feeding habit, have been largely investigated for microplastic uptake and physiological effects; however, most studies were focused on small polystyrene spherical particles, while less data are available for fibers and other polymers. In this work, Polyester textile microfibers (PES-MF) (220 ± 200 µm; range 12 µm –2.9 mm) were utilized. These MF have been previously characterized and investigated for their uptake and effects in soil invertebrates. Data are reported on the effects of PES-MF in the marine invertebrate model, the mussel Mytilus galloprovincialis. In this species, long term exposure to MF has been recently shown to affect a variety of biomarker responses at the tissue level. In vitro short-term experiments were carried out in hemocytes and immune parameters were evaluated. The results show strong physical interactions of hemocytes with PES-MF leading to lysosomal stress and general induction of immune responses (1 to 4h; 50 and 100 µg /mL). In in vivo exposure (96 h, 100 µg/L), the effects of PES-MF were evaluated by a battery of biomarkers: lysosomal responses, immune parameters, and antioxidant enzyme activities. In vivo exposure to PES microfibers did not affect mussel survival; however, changes in immune parameters and other biomarkers were observed. The results highlight the mussel immune system as a sensitive target also for MP fibers, and strengthen the suitable use of both in vitro and in vivo experiments for studying the adverse effects of different types of MPs in marine invertebrates.

Chlamydomonas physiological responses to salinity stress
PRESENTER: Emma Bazzani

ABSTRACT. Salinity is among the main drivers affecting the growth and distribution of photosynthetic organisms in their natural environment, especially in coastal areas, where run-off, rivers and land use have greater impact. In addition to their natural variability, global and regional salinity patterns are rapidly changing as a consequence of human activities, with strong implications on the distribution and composition of microalgal communities, which are the base of the food web. As salinity is potentially a good indicator for detecting anthropogenic change in the next few decades, we performed a comprehensive experiment exposing a microalga from the genus Chlamydomonas (Chlamydomonas sp CCMP225) to different concentrations of sea salts. The evaluation of the microalgal responses to this stressor will shed light on the morphological and physiological effects of salinity stress on green algae, allowing us to gain some insights on the possible effects of climate change on this group of organisms. Notably, our strain appeared to be exceptionally resistant to this kind of stress. In fact, even though the growth was considerably reduced under high salinity, the photosynthetic parameters were not substantially affected. Further studies will elucidate the molecular mechanisms underlying its plasticity. These results could help to understand why some organisms are more favored than others under anthropogenic stressful conditions, and which characteristics give them extreme resilience and adaptability.

Assessing microplastics presence and effects in edible bivalves from the Lagoon of Venice

ABSTRACT. Microplastics (MPs) are an increasing presence in all kinds of environments worldwide with oceans as the ultimate sink. Therefore, marine organisms are continuously exposed to MPs, with potentially negative consequences which need a more in-depth evaluation. In this context, the present project was aimed at assessing the potential risk for the food web and human consumption of filter-feeding bivalves contaminated with MPs. The first aim of the project was to investigate the distribution of MPs in the Lagoon of Venice, using two edible bivalves, the mussel Mytilus galloprovincialis and the clam Ruditapes philippinarum, as sentinel organisms. To assess the presence of MPs extensively, mussels and clams were sampled in 19 and 8 sites, respectively, covering the entire area of interest. In mussels, the highest number of MPs/individual were found in the areas characterized by high anthropogenic influence, compared to more remote areas of the Lagoon. Higher MPs content was found in larger sized animals, suggesting bioaccumulation. However, the ratio MPs number/wet weight was higher in smaller animals. This result highlights the need to properly consider the animal size in MPs quantification studies. The second aim of the project was to age MPs under natural environmental conditions and to check changes in the biofilm growing on them. After 8 months of MP ageing, mussels were fed with weathered MPs. After 7 days of exposure, mussels were sampled to assess MP uptake, as well as animals’ immunological and antioxidant defences, histopathological lesions and microbiota alterations. The first results indicate alterations in immunological status and antioxidant enzyme activities in mussel haemolymph, gills and digestive gland.

Seasonal variability of gene transcription, digestive gland microbiome and shell properties in Mytilus galloprovincialis farmed along the North-Western Adriatic Sea (Italy)
PRESENTER: Letizia Iuffrida

ABSTRACT. Aquaculture of bivalve mollusks is facing the effects of climate changes and widespread sources of chemical pollutants. Being filter feeders, bivalves are particularly exposed to contaminants within their food chain, with effects spanning from reducing growth rates and shell calcification efficiency, to altering reproduction, increasing infections and diseases. Changes of environmental parameters imposed by global warming may act synergistically altering environmental fate and enhancing bioavailability of pollutants or exacerbating their physiological effects on organisms. Taken together, these phenomena may have relevant impacts on several economic and social aspects related to marine food production, from productivity and quality to safety and sustainability. This study assesses seasonal trends of physiological responses in terms of metabolism, cytoprotection/detoxification, neuroendocrine control, and biomineralization in farmed Mediterranean mussels (Mytilus galloprovincialis) along the Italian coasts of the North Adriatic Sea with the final aim to provide baseline information on animal physiological trends under farming conditions and likely forecast potential detrimental impacts of climatological/environmental changes in the study area. Sampling campaign was performed across 8 months (encompassing three seasons) from a farm located in the North-western coast of the Adriatic Sea. Gene transcriptional profiles were assessed, along with digestive gland microbiome and shell structural parameters. All gene products showed complex transcriptional patterns. In fact, seasonal variations were observed. Seasonal differences were also highlighted in the digestive gland microbiome composition and structural properties of the shells. These findings suggest the influence of the environmental variables on all the investigated parameters.

Exposure to nano and microplastic triggers a cytotoxic effect on the gills tissue of Mytilus galloprovincialis
PRESENTER: Rossana Romano

ABSTRACT. Microplastic contamination in the marine environment represents one of the most contemporary pollution problems. Different factors, physical and chemicals such as temperature, wave action, and photo-oxidation drive the fragmentation process. The reduced particle size and their increased bioavailability (1) have determined microplastic accumulation in a large variety of organisms, at different trophic levels (2). In our studies, we investigated the cytotoxic effects of nano and microplastics in Mytilus galloprovincialis, a model organism used in the last decades as a good bioindicator. For this purpose, animals were exposed to polystyrene (5 or 0,1 µm) for 1, 3, or 11 days. Time points and concentrations were used according to the literature and a pilot study (3). At sampling, gills, gonads, digestive glands, and byssus were fixed and processed for light microscopy. The effects were investigated by morphological analysis. Preliminary results show that both micro and nano plastics interfere with gills tissue morphology at early and late time points, on the epithelium and mucus cells. In deep, significant alterations are observed in the septum and lamellae organization, and granules and infiltration are detected. Mucus cells are differently distributed and increased in number, especially after nano plastics exposure. Immunocytochemistry investigations by PCNA, demonstrated an increased number of positive cells in the tissues after treatment compared to the control. We can conclude that polystyrene induces tissue damage in the gills of the mussels altering their function. Future investigation will clarify how the treatments have affected cellular stress response and the consequence on fitness and reproductive success. This study provides a clue for future investigation finalized to ameliorate risk assessment for marine plastic pollution. 1. Andrady, Mar. Poll. Bull. 2011 62 2. Browne et al., Cur. Bio. 2013 23 3. Vroom et al., Env. poll. 2017 231

Comparing the effects of four antidepressants and two metabolites on early life stages of the mussel, Mytilus galloprovincialis
PRESENTER: Elena Fabbri

ABSTRACT. The occurrence of pharmaceutical residues in aquatic ecosystems represents a cause of concern, especially related to the issue of (neuro)endocrine disruption by environmental contaminants. This particularly applies to antidepressants, e.g. fluoxetine, FLX; sertraline, SERT; citalopram, CITA (selective serotonin reuptake inhibitors) and venlafaxine, VEX (serotonin-norepinephrine re-uptake inhibitor). Their mode of action is modulation of neurotransmitter permanence in the synaptic cleft, in particular serotonin which has a pivotal role in molluscs. Antidepressant concentrations from µg/L in inland waters to ng/L in marine waters have been reported, with values > 500 ng/L in urban estuaries. Striking low-dose effects of FLX were reported on marine invertebrates. Here we report the effects of 4 antidepressants (FLX, SERT, CITA, VEX) and 2 metabolites (Norfluoxetine, NF; O-Desmethylvenlafaxine, ODV), analysed in parallel on embryo/larval stages of Mytilus galloprovincialis. The endpoints include fertilization toxicity test, larval motility/mortality, and embryotoxicity assessed within a 0.5 - 500 ng/l concentration range of the compounds. After exposure, significant effects were observed on embryo development and fertilization, while the effects on motility and mortality of mussel larvae were low. In particular, 48 h post fertilization, D-veliger embryos were found to develop abnormally after exposure to NF (5 – 500 ng/l), SERT (25 – 500 ng/l), FLX (100 – 500 ng/l) and CITA (500 ng/l). Differently, VEX and ODV did not influence the normal development of embryos. Fertilization toxicity test showed significant effects by SERT (10 - 500 ng/l) and ODV (25 - 500 ng/l); FLX and NF also had significant effects at higher concentrations (100 – 500 ng/l). Fertilization success did not appear to be affected by either VEX or CITA. In conclusion, antidepressants and their metabolites may affect marine life early development at concentrations occurring in marine water.

Ecotoxicological effects on polychaetes (Hediste diversicolor) exposed to environmentally realistic concentrations of nickel nanoparticles
PRESENTER: Eduardo Motta

ABSTRACT. The objective of this study was to evaluate the ecotoxicological chronic (28 days) effects of nickel nanoparticles (NiNP) at environmentally realistic concentrations (0.05 mg/L; 0.5 mg/L; 5 mg/L), including a negative control, in Hediste diversicolor. Antioxidant defense enzymes (Catalase, CAT), phase II metabolic detoxification (Glutathione S-Transferases, GSTs) and lipid peroxidation (thiobarbituric acid reactive species, TBARS) were evaluated. GST values were significantly higher in the exposed groups compared with the control. However, when it comes to the enzymatic evaluation for CAT, only the two groups that received the highest NiNP doses presented higher activities compared to control individuals. The hereby results suggest that the NiNP promoted biochemical changes in the polychaete Hedistes diversicolor. However, the mechanisms behind these effects needs to be further studied.

Ecotoxicological effects on fish (Salmo trutta fario) exposed to environmentally realistic concentrations of omeprazol
PRESENTER: Nazaré Coelho

ABSTRACT. Omeprazole is a widely prescribed pharmaceutical drug that acts as a proton inhibitor pump. It could enter the aquatic compartment being inappropriately flushed down the toilet. However, studies regarding the post-exposure ecotoxicological effects in aquatic organisms are scarce. In this study, Salmo trutta fario were chronically exposed (28 days) to environmentally realistic concentrations of parental omeprazole (0.01, 0.1, and 1.0 µg/L), including a negative (unexposed) control group. Fish gills and liver were assessed in terms of antioxidant defense enzymes (Catalase, CAT), phase II metabolic detoxification (Glutathione S-Transferases, GSTs) and lipid peroxidation (thiobarbituric acid reactive species, TBARS). Results showed some deleterious effects. However, further studies namely by using additional biomarkers are need to elucidate the mechanism behind these effects.

Funding: This work was supported by supported by the Portuguese Foundation for Science and Technology (UID/Multi/04423/2019), by the European Regional Development Fund (PT2020) and by Ocean 3R (Norte 2020, Portugal 2020 and European Regional Development Fund).

Effect of functional bread with resveratrol on diabetes-induced cardiac remodeling

ABSTRACT. The World Health Organization has a global target to halt the rise of diabetes by 2025 and functional bread with resveratrol (RSV) could be a novel approach. Evidence shows that RSV can reduce cardiac oxidative stress, mitochondrial dysfunction, and myocardial fibrosis induced by the elevated serum glucose concentrations seen in diabetes. This study aimed to evaluate the systemic effect of this functional bread to prevent diabetes complications in vivo. Forty-seven male Sprague-Dawley rats (4 weeks old) were divided in control group with plain bread (CB), control with functional bread (CBR), diabetic with plain bread (DB) and diabetic with functional bread (DBR). Plain bread and functional bread diet (10 mg RSV/Kg body weight) were given to rat groups after diabetes induction with streptozotocin (40 mg/Kg) in diabetic groups. After four weeks of diet, the animals were euthanized under approved procedures. Anthropometric parameters were measured, and heart was homogenized and subject to western-blot analysis of markers of inflammation, oxidative stress and metabolism. The serum collected was used for glucose (Glc) quantification (Cormay kit) and immunoblot assays of inflammation and oxidative stress markers. Serum Glc levels confirmed the animals' diabetic state from diabetes-induced groups. The anthropometric parameters revealed that diabetic animals hold the lowest body weight and smaller hearts than non-diabetic ones. Also, this study had a low mortality rate of 6.25%. Serum immunoblot assays showed no signs of inflammation and oxidative stress in all animals. In the cardiac tissue of diabetic rats, a lower sensibility to insulin was seen, given by the increased content of electron transfer flavoprotein dehydrogenase, which support a higher reliance on fatty acids oxidation for energetic purposes, characteristic of diabetic heart, and alterations in intercellular communication, considering the increase of the gap junction protein conexin 43 in diabetic animals.

Brain biochemical alterations in wild fish (Chelon auratus) pinpointed a higher vulnerability to mercury neurotoxicity in winter than summer at a contaminated coastal lagoon
PRESENTER: Fátima Brandão

ABSTRACT. Fish brain demonstrated to be a target organ for mercury (Hg) bioaccumulation and its neurotoxicity in fish living at Hg contaminated areas has been evidenced. This study aims to be a step forward on the knowledge of Hg neurotoxicity in an estuarine fish species (Chelon auratus) by putting together, Hg bioaccumulation, oxidative stress related endpoints (antioxidants and peroxidative damage) and neurotransmission parameters [glutamine synthetase (GS) and acetylcholinesterase (AChE)] to better understand the Hg neurotoxicity-associated mechanisms in estuarine fish under realistic field scenarios. This approach was conducted in two sites of the Aveiro lagoon: (i) Largo do Laranjo (LAR) located in an Hg contaminated area, and (ii) São Jacinto (SJ) selected as reference site; and replicated in two contrasting seasons (winter and summer). Inorganic Hg (iHg) and methylmercury (MeHg) accumulation in fish brain was higher at LAR than SJ in both seasons, reflecting environmental spatial differences of water column and surface sediments. Besides, a winter-summer variation was found for LAR site, with winter levels of total Hg (tHg) and MeHg in the brain being higher than those accumulated in summer. Regarding oxidative stress and neurotransmission parameters, fish caught at LAR in winter showed an increase of superoxide dismutase (SOD) activity and a decreased GS activity relatively to the SJ specimens, suggesting that exposure to Hg forms may be responsible by the enhancement of brain antioxidant defenses and alterations on the glutamate homeostasis. Moreover, higher levels of total glutathione were recorded in brain of fish from LAR in summer. Current data pinpointed a higher vulnerability of fish brain to Hg neurotoxicity in winter than summer at a Hg contaminated coastal lagoon. This neurotoxicant can be accumulated in brain, leading to alterations in the cellular oxidative stress protection and neurotransmission, possibly with further repercussions on fish fitness.

Exploring the interference of a lipophilic toxin and an extensively used organic sunscreen in the clam (Donax trunculus) by a biochemical approach
PRESENTER: Eugenia Toso

ABSTRACT. The clam Donax trunculus is of high economic value in the South of Europe, being also an emblematic species in some regions. Unfortunately, its capture is often interdicted due to the presence of high levels of okadaic acid (OA), a lipophilic toxin, in its edible tissues during blooms of harmful algae. Specifically in Portugal, the interdiction time of D. trunculus captures has been increasing in the last years, namely in the summer months. This study explores the hypothesis that the exposure of D. trunculus to water contaminants (namely a common sunscreen ingredient in hygiene and personal care products – oxybenzone, also known as benzophenone 3) could delay the bivalve metabolization of OA, contributing to enhancing the interdiction time. In fact, in the summer months, the occurrence of oxybenzone in coastal waters should increase following the frequent application of sunscreen products by beach users. In the present work, an exposure experiment of D. trunculus to realistic levels of OA (10 ng/L) and oxybenzone (5 μg/L) (individual exposure and in combination) for 7 days was performed. Clams were sampled for the quantification of OA concentrations in the edible tissues upon 3 (E3) and 7 (E7) days of exposure, and after 7 days of post-exposure (PE7), as well as for the determination of biochemical parameters related to biotransformation (EROD and GST), glutathione-related enzymes (GR, GPx) and peroxidative damage (LPO). A significant increase of GPx was found in the clams exposed to OA plus oxybenzone, in relation to control organisms at E7, while no changes were found upon exposure to each compound individually. Differently, oxybenzone alone triggered a decrease of GPx in the post-exposure time (PE7). Moreover, the combination of both compounds also elicited a significant decrease of GR and an enhancement of LPO in the post-exposure time, suggesting long-lasting effects of both compounds when in combination.

Neuroendocrine pathways at risk? Simvastatin induces inter and transgenerational disruption in the keystone amphipod Gammarus locusta
PRESENTER: Nélson Alves

ABSTRACT. There are several gaps regarding the effects that parental exposure (F0) to environmental conditions exerts on indirectly exposed (F1 and F2) or truly non-exposed (F3) generations - intergenerational and transgenerational, respectively. Pharmaceuticals are being increasingly reported as modulators of biological processes of non-target organisms in several exposed and non-exposed generations, as is the case of simvastatin, one of the most widely prescribed human pharmaceuticals for the primary treatment of hypercholesterolemia. Our previous research with simvastatin reported marked reproductive and growth effects in the keystone crustacean Gammarus locusta after a direct, inter and transgenerational exposure to environmentally relevant concentrations of this pharmaceutical. Therefore, considering our previous findings and the involvement of neuroendocrine pathways in reproductive processes, the present study performed a comprehensive RNA-seq analysis with G. locusta that assessed the inter and transgenerational (F1 and F3) molecular changes of genes/pathways related with the regulation of the endocrine system. we demonstrate that simvastatin, at environmentally relevant concentrations, has significant inter and transgenerational effects in five key signaling pathways involved in crustaceans’ neuroendocrine regulation, i.e., Ecdysteroids, Catecholamines, NO/cGMP/PKG, GABAergic and Cholinergic signaling pathways. These findings are critical to improve the hazard and risk assessment of environmental contaminants such as simvastatin, highlighting that neuroendocrine regulation should be implemented in this evaluation, including the effects perpetuated throughout the non-exposed generations.

Impact of vermicomposting of animal manure/sewage sludge mixtures in Eisenia fetida
PRESENTER: José Gonçalves

ABSTRACT. Sewage sludge (SS) is an important source of nutrients and organic matter (OM) when applied to soils. However, the stability of OM and/or the presence of contaminants may limit its use in agriculture. The vermicomposting bioprocess, although an alternative to overcome these limitations, may affect the earthworms. Defining the most suitable conditions for earthworms’ performance will contribute to the success of SS vermicomposting. The present work aims to evaluate the impact of manure/SS mixtures in Eisenia fetida including metals bioaccumulation. Seven substrates were considered: M1, M2, M3, M4 plus M1S, M2S, M3S, the last 3 consisting of M1, M2 and M3 supplemented with 25% of SS. M1 to M4 were pre-composted mixtures of sewage sludge (SS), horse manure (HM) and rice husk (RH): M1- 45% SS, 45% HM, 10% RH; M2- 35% SS, 55% HM, 10% RH; M3- 25% SS, 65% HM, 10% RH; M4- 100% HM. Earthworms were added to the substrates (29/kg fresh weight) during 31 days. The parameters studied were: earthworm weight variation, mortality, reproduction and metal bioaccumulation factors-BAFs (Cu, Zn, Ni, Cd, Pb, Cr). The results showed 100% of mortality in M1, M1S and M2S after 31 days of vermicomposting. In treatments with 0% mortality, mean body weight, decreased in M2 and M3, and increased in M4 and M3S between 0 and 31 days. Cocoon production was observed in M4 and M3S: 11.22 and 0.07 cocoons/earthworm, respectively. BAFs increased with the decrease of SS in the mixtures for all the metals, except Cr where M4 reached the lowest value. BAFs>1 were observed for Cd in all the treatments and for Zn in M4. Sewage sludge had a significant impact on earthworms’ survival and reproduction and negatively affected metal bioaccumulation capacity of earthworms, suggesting that other SS contaminants/factors are negatively affecting earthworms.

Physiological effects of environmental chemicals on colour change in common cuttlefish: ex-vivo quantification on skin explants

ABSTRACT. Body colour change is a widespread ability among animals associated with a series of functions: thermoregulation, UV protection, camouflage or communication. Cephalopods provide well studied models for their colour change abilities known to be particularly rapid and finely controlled by nervous pathways. Their skin is a complex organ that includes a variety of chromatophores (yellow-, red- and dark-pigmented cells) laying on top of light-reflecting leucophores and iridophores. Dynamic colour changes mainly involve the mechanical action of chromatophore-associated neuromuscular structures, resulting in the expansion or contraction of pigmented organelles. Since many contaminants present in the environment can interfere with the functions of the nervous system, the measurement of colour changes is currently emerging as a promising toxicological endpoint. In practice, colour changes can be observed and measured on whole animals as part of behaviours, but also at the tissue and cellular levels. Here we focus on the quantification of chromatophore activity from skin explants of the common cuttlefish (Sepia officinalis) in response to topical application of contaminants. A bioassay is presented that takes advantage of a new method for automated analysis, based on deep learning. Its performances are illustrated by the analysis of responses to: i) glutamate and serotonin as positive control drugs (neurotransmitters known to control the chromatophore expansion and contraction); ii) antidepressants (Serotonin Selective or Serotonin Norepinephrine Reuptake Inhibitors), and iii) putative endocrine disrupting chemicals.

Experimental test of physiological effects of marine heatwave in the canopy-forming macroalgae Gongolaria barbata
PRESENTER: Carmen Arena

ABSTRACT. Among the occurring climate changes related to the use of fossil fuels, seawater warming and marine heatwaves (MHWs) are affecting the integrity of coastal marine habitats including macroalgal forests of the genus Cystoseira sensu latu. Understanding the resilience and potential for adaptation of marine habitat forming species to future ocean warming becomes pivotal for predicting future changes, improving present conservation and restoration strategies. In this study, we investigated the physiological effects of short vs long MHWs on recruits of Gongolaria barbata collected from the Lagoon La Strea (Porto Cesareo, Puglia), by means of a thermos-tolerance experiment. Photosynthetic pigment content, PSII maximal photochemical efficiency (Fv/Fm) and amount of antioxidant compounds were measured. Our results demonstrate that the antioxidant soluble pool, namely total antioxidant activity polyphenols, tannins, and flavonoids, is strongly modulated by the length of the thermal stress period. In particular, the polyphenol content and antioxidant capacity increased in thalli exposed to long-term stress compared to control and short-term exposures to high temperature. Furthermore, from a physiological point of view, the higher chlorophyll and carotenoid content, as well as the higher value of Fv/Fm ratio in recruits exposed to long-term stress likely indicate an acclimation of thalli to the new environmental condition and increased tolerance of G. barbata to thermal stress.

Coelomocytes and riboflavin concentrations in Eisenia fetida exposed to WWTP sludges
PRESENTER: Cláudia Pinto

ABSTRACT. In earthworms, coelomocytes constitute the first line defense, varying in number and type in the course of infections or exposure to contaminants, with these cells being naturally autofluorescent. Riboflavin is the main fluorophore present and known to be responsive to environmental factors. The aim of this work was to evaluate WWTP sludges toxicity, based on the concentrations of coelomocytes and riboflavin. Earthworms (n=12/replicate) were kept 30 and 60 days in substrates with WWTP sludges (M1-45%; M2-35%;M3- 25%), horse manure (M1-45%, M2-55%; M3-65%) and rice husk(10%.) Control earthworms were kept in horse manure. For coelomic fluid harvesting, earthworms were stimulated for 15 sec with a 4,5 V electric current in PBS extrusion buffer. For coelomocyte counting, the extrusion solution was fixed in 2% formalin and cells concentration was determined in a hemocytometer. The remaining extrusion solution was used for riboflavin concentration determination, starting with an ultrasound cell lysis, followed by spectrofluorometer analysis. At the end of 30 days, the M1 group presented 100% mortality. The concentration of coelomocytes was significantly lower in earthworms maintained on substrates with sludge compared to control, with the greatest decrease being observed in M2. Comparing the two collection times, there was a decrease in coelomocytes concentration in the control and in M3 groups. Regarding riboflavin concentration, it was evident a decrease, related to the amount of sludge, at the end of 60 days. Interestingly, riboflavin concentration in the control increased with time. Differences observed in control could be related to the vermicomposting process, namely acidification of the substrates, mineralization of organic matter and changes in the bioavailability of the present metals. The results show that the presence of WWTP sludges on the substrates are toxic to earthworms, resulting in a reduction in both coelomocytes and riboflavin concentrations.

Vertical bars vs. horizontal stripes: the Mediterranean banded killifish Aphanius fasciatus (Valenciennes, 1821) (Teleostei: Cyprinodontidae) as a novel euryhaline fish model for comparative, environmental and ecological physiology

ABSTRACT. Aphanius fasciatus is a small dimorphic fish found in large-sized populations in coastal brackish waters of the central-eastern Mediterranean Sea. Despite sporadic presence in coastal marine waters, most common habitats are transitional waters, and fresh and brackish waters as weedy ponds and ditches. This contributes to restrict gene flow, that may occur between adjacent populations. Due to the virtually complete isolation of brackish water habitats and selective environmental features, A. fasciatus populations diverge. A. fasciatus tolerate harsh environmental conditions, including high temperature and salinity and low oxygen levels. Reproduction takes place from April to September with life history traits including external fertilization, demersal eggs deposited on benthic vegetation, short generation time (~3.5 yrs), high reproductive rate and rapid population turnover. Diet is made of invertebrates, mainly crustaceans and insect larvae. A. fasciatus is typified by habitat fidelity in its adult phase, and hatchlings show a lifestyle like that of adults. A. fasciatus look as suitable models to study growth and feeding ecology, population dynamics, morphological polymorphism, genetic diversity, phenotype-genotype relations, micro/macroevolutionary processes, evolution of physiological adaptations, reproductive behavior and sexual selection, and as effective sentinels in coastal ecosystems. To find out if A. fasciatus may emerge as a euryhaline (in field & lab) fish model, we started investigating its biology and evaluating its capacity of being kept in captivity. Few hundred records are based in Entrez so far. Compared to Cyprinodontiformes with genome and toolbox available (e.g., Nothobranchius furzeri), the possibility of starting a Mediterranean killifish genome sequencing project is also discussed. Supported by ‘Monitoraggio di Siti, Specie e Habitat, Natura 2000 in Puglia (MoSSHa)’, POR-POC PUGLIA 2014-2020. ASSE VI - AZIONE 6.5 Sub Azione 6.5.a (Project 17).