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

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

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

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

Differential HSP70 gene expression in the Mediterranean mussel exposed to various stressors

HSP70 gene expression was studied by quantitative RT-PCR after cloning and sequencing of two different HSP70 gene fragments from the digestive gland of Mytilus galloprovincialis, called MgHSP70 and MgHSC70. Heat shock (1h at 35 degrees C) caused rapid induction of MgHSP70, while no change was observed for MgHSC70. Hg(2+) (150mug/L for different time periods) significantly induced MgHSP70 expression that reached maximum levels after 24h, decreasing thereafter. MgHSC70 expression was inhibited after 1 day and induced after a 6-day exposure to Hg(2+). A 1-week exposure to Cr(6+) (1, 10, and 50ng/L) induced and inhibited MgHSC70 and MgHSP70 transcript levels, respectively. MgHSC70 and MgHSP70 appear to play different roles in cell protection; the former is induced after acute stress and/or during the earlier phase of the response while the latter is induced by chronic stress. The present results provide new insights into mechanisms used by mussels to adapt to stressful environments

PKA-mediated transcriptional regulation of the Mediterranean mussel ABCB1 gene involved in the Multixenobiotic Resistance system

Marine mussels display a high capability of adaptation to contaminated environments, due to the activation of different cytoprotective responses. The Multixenobiotic Resistance system (MXR) represents a broadly distributed physiological mechanism acting as a first-tier defense against xenobiotics. Scarce information is available about the regulatory features of the MXR-related proteins in bivalves, including the P-glycoprotein (Pgp), which is the most studied under an environmental context. Investigations on mammals showed that expression of the ABCB1 gene encoding Pgp is under a cAMP/PKA-mediated regulation. In this study haemocytes from the mussel Mytilus galloprovincialis were selected as a cell model to assess whether a similar evolutionary-conserved regulatory framework exists also in bivalves. The experimental data clearly showed that PKA is directly involved in the regulation of ABCB1 gene transcription. Indeed dB-cAMP, the specific PKA activator, increased the enzyme activity and simultaneously up-regulated the mussel ABCB1 gene expression, while H89, the specific PKA inhibitor, counteracted the stimulatory effects of dB-cAMP on both endpoints. Moreover, serotonin, a physiological agonist acting on mussel haemocytes, induced the concerted down-regulation of both PKA activity and ABCB1 gene expression, while noradrenaline was ineffective. The serotonin effect was abolished by propranolol, which proved able to block 5HT receptors. These data provide the first direct demonstration that the mussel ABCB1 gene is under a PKA transcriptional regulation, and also that physiological modulators of the cAMP/PKA system can directly affect the ability of mussels to cope with environmental pollutants