Impact of bisphenol A (BPA) on early embryo development in the marine mussel Mytilus galloprovincialis: Effects on gene transcription.

Bisphenol A (BPA), a monomer used in plastic manufacturing, is weakly estrogenic and a potential endocrine disruptor in mammals. Although it degrades quickly, it is pseudo-persistent in the environment because of continual inputs, with reported concentrations in aquatic environments between 0.0005 and 12 \u3bcg/L. BPA represents a potential concern for aquatic ecosystems, as shown by its reproductive and developmental effects in aquatic vertebrates. In invertebrates, endocrine-related effects of BPA were observed in different species and experimental conditions, with often conflicting results, indicating that the sensitivity to this compound can vary considerably among related taxa. In the marine mussel Mytilus galloprovincialis BPA was recently shown to affect early development at environmental concentrations. In this work, the possible effects of BPA on mussel embryos were investigated at the molecular level by evaluating transcription of 13 genes, selected on the basis of their biological functions in adult mussels. Gene expression was first evaluated in trocophorae and D-veligers (24 and 48 h post fertilization) grown in physiological conditions, in comparison with unfertilized eggs. Basal expressions showed a general up-regulation during development, with distinct transcript levels in trocophorae and D-veligers. Exposure of fertilized eggs to BPA (10 \u3bcg/L) induced a general upregulation at 24 h pf, followed by down regulation at 48 h pf. Mytilus Estrogen Receptors, serotonin receptor and genes involved in biomineralization (Carbonic Anydrase and Extrapallial Protein) were the most affected by BPA exposure. At 48 h pf, changes in gene expression were associated with irregularities in shell formation, as shown by scanning electron microscopy (SEM), indicating that the formation of the first shelled embryo, a key step in mussel development, represents a sensitive target for BPA. Similar results were obtained with the natural estrogen 17\u3b2-estradiol. The results demonstrate that BPA and E2 can affect Mytilus early development through dysregulation of gene transcription

Phenotypical and molecular changes induced by carbamazepine and propranolol on larval stages of Mytilus galloprovincialis

The possible impact of carbamazepine (CBZ) and propranolol (PROP), two widespread pharmaceuticals in the aquatic environment, were investigated on morphology and gene transcription of early larvae of Mytilus galloprovincialis. Pharmaceuticals were first tested in a wide concentration range (from 0.01 to 1000 a) through the 48-hpf embryotoxicity assay. The results showed that both compounds significantly affected embryo development from environmental concentrations. Although similar EC50 were obtained, (congruent to 1 mu g/L) CBZ induced a progressive increase in embryo malformations, whereas PROP apparently showed greater impacts in terms of arrested development and embryo mortality at higher concentrations (>10 mu g/L). Transcriptional analyses of 17 genes involved in different physiological functions in mussels and/or in their response to environmental contaminants, were performed at 24 and 48 h pf at two selected concentrations of CBZ and PROP (0.01 and 1 mu g/L). Both compounds induced down-regulation of shell-specific and neuroendocrine related transcripts, while distinct effects were observed on antioxidant, lysosomal, and immune-related transcripts, also depending on the larval stage investigated. The results demonstrate that CBZ and PROP can affect development and gene transcription in mussel early larvae at environmental concentrations. (C) 2019 Elsevier Ltd. All rights reserved

Long-term effects of an informal education program on tourist environmental perception

Tourism is one of the most important economic sectors worldwide, with significant overarching impact on the environment, including negative effects caused by tourist inappropriate behavior while on vacation. By providing informal educational activities, tourism also has an educative role that leads to positive learning outcomes and beneficial environmental effects. Here we present the short- and long-term outcomes of a project for environmental education (Glocal Education) carried out in three travel destinations, aimed at promoting sustainability variables (knowledge, attitude, and awareness) in participating tourists. Since psychological components can affect learning outcomes, we also considered tourist satisfaction in participating in the project and identification with its values, as well as the intention to travel with the hosting tour operator again in the future. Tourists were asked to complete evaluation questionnaires three times: before Glocal Education activities, right after activities (i.e., while still on vacation), and after at least one year from initial project participation. Short- and long-term learning outcomes were tested, and possible relations between these variables and psychological components (satisfaction, identification, and intention) of the learning experience were verified. Overall, knowledge, attitude and awareness increased in the short term, while in the long term, knowledge and attitude decreased, and awareness remained constant. In most cases, psychological components showed positive relation with sustainability variables, which suggested their important role in structuring and carrying out environmental education activities. This study suggests that informal environmental education activities can be advantageous for tourism stakeholders in terms of customer loyalty. Such activities can contribute to enhance environment literacy, by allowing tourists to observe the environmental impact caused by human activity, and understand how their day-to-day actions, even if small, might help address some of the current concerns for environmental conservation

Acclimatization of a coral-dinoflagellate mutualism at a CO2 vent

Ocean acidification caused by shifts in ocean carbonate chemistry resulting from increased atmospheric CO2 concentrations is threatening many calcifying organisms, including corals. Here we assessed autotrophy vs heterotrophy shifts in the Mediterranean zooxanthellate scleractinian coral Balanophyllia europaea acclimatized to low pH/high pCO2 conditions at a CO2 vent off Panarea Island (Italy). Dinoflagellate endosymbiont densities were higher at lowest pH Sites where changes in the distribution of distinct haplotypes of a host-specific symbiont species, Philozoon balanophyllum, were observed. An increase in symbiont C/N ratios was observed at low pH, likely as a result of increased C fixation by higher symbiont cell densities. δ13C values of the symbionts and host tissue reached similar values at the lowest pH Site, suggesting an increased influence of autotrophy with increasing acidification. Host tissue δ15N values of 0‰ strongly suggest that diazotroph N2 fixation is occurring within the coral tissue/mucus at the low pH Sites, likely explaining the decrease in host tissue C/N ratios with acidification. Overall, our findings show an acclimatization of this coral dinoflagellate mutualism through trophic adjustment and symbiont haplotype differences with increasing acidification, highlighting that some corals are capable of acclimatizing to ocean acidification predicted under end-of-century scenarios

Effect of high-frequency electromagnetic fields on trophoblastic connexins.

Connexins (Cx) are membrane proteins able to influence trophoblast functions. Here we investigated the effect of high-frequency electromagnetic fields (HF-EMF) on Cx expression and localization in extravillous trophoblast cell line HTR-8/SVneo. We also analysed cell ultrastructural changes induced by HF-EMF exposure. Samples were exposed to pulse-modulated 1817 MHz sinusoidal waves (GSM-217 Hz; 1 h: SAR of 2 W/kg). Cx mRNA expression was assessed through semi-quantitative RT-PCR, protein expression by Western blotting, protein localization by indirect immunoflorescence, cell ultrastructure using electron microscopy. HF-EMF exposure significantly and selectively increased Cx40 and Cx43, without altering protein expression. Nevertheless, Cx40 and Cx43 lost their punctuate fluorescence within the cell membrane, becoming diffuse after HF-EMF exposure. Electron microscopy evidenced a sharp decrease in intercellular gap junction-like structures. This study is the first to indicate that exposure of extravillous trophoblast to GSM-217 Hz signals can modify Cx gene expression, Cx protein localization and cellular ultrastructure

Concentration-Dependent, Size-Independent Toxicity of Citrate Capped AuNPs in Drosophila melanogaster

The expected potential benefits promised by nanotechnology in various fields have led to a rapid increase of the presence of engineered nanomaterials in a high number of commercial goods. This is generating increasing questions about possible risks for human health and environment, due to the lack of an in-depth assessment of the physical/chemical factors responsible for their toxic effects. In this work, we evaluated the toxicity of monodisperse citrate-capped gold nanoparticles (AuNPs) of different sizes (5, 15, 40, and 80 nm) in the model organism Drosophila melanogaster, upon ingestion. To properly evaluate and distinguish the possible dose- and/or size-dependent toxicity of the AuNPs, we performed a thorough assessment of their biological effects, using two different dose-metrics. In the first approach, we kept constant the total surface area of the differently sized AuNPs (Total Exposed Surface area approach, TES), while, in the second approach, we used the same number concentration of the four different sizes of AuNPs (Total Number of Nanoparticles approach, TNN). We observed a significant AuNPs-induced toxicity in vivo, namely a strong reduction of Drosophila lifespan and fertility performance, presence of DNA fragmentation, as well as a significant modification in the expression levels of genes involved in stress responses, DNA damage recognition and apoptosis pathway. Interestingly, we found that, within the investigated experimental conditions, the toxic effects in the exposed organisms were directly related to the concentration of the AuNPs administered, irrespective of their size

Ocean Acidification at High Latitudes: Potential Effects on Functioning of the Antarctic Bivalve Laternula elliptica

Ocean acidification is a well recognised threat to marine ecosystems. High latitude regions are predicted to be particularly affected due to cold waters and naturally low carbonate saturation levels. This is of concern for organisms utilising calcium carbonate (CaCO3) to generate shells or skeletons. Studies of potential effects of future levels of pCO2 on high latitude calcifiers are at present limited, and there is little understanding of their potential to acclimate to these changes. We describe a laboratory experiment to compare physiological and metabolic responses of a key benthic bivalve, Laternula elliptica, at pCO2 levels of their natural environment (430 µatm, pH 7.99; based on field measurements) with those predicted for 2100 (735 µatm, pH 7.78) and glacial levels (187 µatm, pH 8.32). Adult L. elliptica basal metabolism (oxygen consumption rates) and heat shock protein HSP70 gene expression levels increased in response both to lowering and elevation of pH. Expression of chitin synthase (CHS), a key enzyme involved in synthesis of bivalve shells, was significantly up-regulated in individuals at pH 7.78, indicating L. elliptica were working harder to calcify in seawater undersaturated in aragonite (ΩAr = 0.71), the CaCO3 polymorph of which their shells are comprised. The different response variables were influenced by pH in differing ways, highlighting the importance of assessing a variety of factors to determine the likely impact of pH change. In combination, the results indicate a negative effect of ocean acidification on whole-organism functioning of L. elliptica over relatively short terms (weeks-months) that may be energetically difficult to maintain over longer time periods. Importantly, however, the observed changes in L. elliptica CHS gene expression provides evidence for biological control over the shell formation process, which may enable some degree of adaptation or acclimation to future ocean acidification scenarios

Cyclic-AMP Mediated Regulation of ABCB mRNA Expression in Mussel Haemocytes

BACKGROUND: The multixenobiotic resistance system (MXR) allows aquatic organisms to cope with their habitat despite high pollution levels by over-expressing membrane and intracellular transporters, including the P-glycoprotein (Pgp). In mammals transcription of the ABCB1 gene encoding Pgp is under cAMP/PKA-mediated regulation; whether this is true in mollusks is not fully clarified. METHODOLOGYPRINCIPAL FINDINGS: cAMP/PKA regulation and ABCB mRNA expression were assessed in haemocytes from Mediterranean mussels (Mytilus galloprovincialis) exposed in vivo for 1 week to 0.3 ng/L fluoxetine (FX) alone or in combination with 0.3 ng/L propranolol (PROP). FX significantly decreased cAMP levels and PKA activity, and induced ABCB mRNA down-regulation. FX effects were abolished in the presence of PROP. In vitro experiments using haemocytes treated with physiological agonists (noradrenaline and serotonin) and pharmacological modulators (PROP, forskolin, dbcAMP, and H89) of the cAMP/PKA system were performed to obtain clear evidence about the involvement of the signaling pathway in the transcriptional regulation of ABCB. Serotonin (5-HT) decreased cAMP levels, PKA activity and ABCB mRNA expression but increased the mRNA levels for a putative 5-HT1 receptor. Interestingly, 5-HT1 was also over-expressed after in vivo exposures to FX. 5-HT effects were counteracted by PROP. Forskolin and dbcAMP increased PKA activity as well as ABCB mRNA expression; the latter effect was abolished in the presence of the PKA inhibitor H89. CONCLUSIONS: This study provides the first direct evidence for the cAMP/PKA-mediated regulation of ABCB transcription in mussel