Interactive effects of nanoparticles with other contaminants in aquatic organisms: Friend or foe?

none3The increasing production and use of nanoparticles (NPs) will lead to their release into the aquatic environment, posing a potential threat to the health of aquatic organisms. Both in the water phase and in the sediments NPs could mix and interact with other pollutants, such as organic xenobiotics and heavy metals, leading to possible changes in their bioavailability/bioconcentration/toxicity. However, whether these interactive effects may lead to increased harmful effects in marine organisms is largely unknown. In this work, available data mainly obtained on carbon based NPs and n-TiO2, as examples of widespread NPs, in aquatic organisms are reviewed. Moreover, data are summarized on the interactive effects of n-TiO2 with 2,3,7,8-TCDD and Cd(2+), chosen as examples of common and persistent organic and inorganic contaminants, respectively, in the model marine bivalve Mytilus. The results reveal complex and often unexpected interactive responses of NPs with other pollutants, depending on type of contaminant and the endpoint measured, as well as differences in bioaccumulation. The results are discussed in relation with data obtained in freshwater organisms. Overall, information available so far indicate that interactive effects of NPs with other contaminants do not necessarily lead to increased toxicity or harmful effects in aquatic organisms.openCanesi, L; Ciacci, C; Balbi, TCanesi, L; Ciacci, C; Balbi,

Temporal variation of Skeletonema community composition from a long-term time series in Narragansett Bay identified using high-throughput DNA sequencing

Phytoplankton species cannot always be identified by their morphology using light microscopy, which makes inferring the ecological and biogeochemical importance of individual species a difficult task. Here, a combination of microscopy and high-throughput DNA sequencing was used to examine morphologically cryptic and pseudo-cryptic species in the diatom genus Skeletonema from the Long-Term Plankton Time Series in Narragansett Bay (NBay), where Skeletonema is ecologically important, comprising up to 99% of microplankton cells in surface waters. The 28S rDNA from mock phytoplankton communities comprising known species was amplified and sequenced using newly developed Skeletonema-specific primers. The relative abundances of species in the sequence data did not match expected abundances, suggesting that 28S copy number can vary greatly, even among closely related diatom species. The 28S rDNA was also amplified from 75 field samples collected from 2008 to 2013. A total of 7 Skeletonema species were identified, including 5 newly detected species from NBay. Skeletonema species composition was highly seasonal and significantly correlated with water temperature. Winter–spring and summer–autumn communities were significantly different and characterized by low and high species richness, respectively. Species abundance during winter–spring was quantified by combining sequence data with light microscopy counts, revealing Skeletonema marinoi as the numerically dominant species during the winter–spring bloom. Seasonal variation in Skeletonema composition suggests that, although morphologically similar, species in this genus are likely adapted to different environmental conditions, raising the possibility that species composition of this important bloom-forming genus may shift as water temperatures in NBay increase due to anthropogenic influences

Characterization of the main steps in first shell formation in Mytilus galloprovincialis: possible role of tyrosinase

Bivalve biomineralization is a highly complex and organized process, involving several molecular components identified in adults and larval stages. However, information is still scarce on the ontogeny of the organic matrix before calcification occurs. In this work, first shell formation was investigated in the mussel Mytilus galloprovincialis. The time course of organic matrix and CaCO3 deposition were followed at close times post fertilization (24, 26, 29, 32, 48 h) by calcofluor and calcein staining, respectively. Both components showed an exponential trend in growth, with a delay between organic matrix and CaCO3 deposition. mRNA levels of genes involved in matrix deposition (chitin synthase; tyrosinase- TYR) and calcification (carbonic anhydrase; extrapallial protein) were quantified by qPCR at 24 and 48 hours post fertilization (hpf) with respect to eggs. All transcripts were upregulated across early development, with TYR showing highest mRNA levels from 24 hpf. TYR transcripts were closely associated with matrix deposition as shown by in situ hybridization. The involvement of tyrosinase activity was supported by data obtained with the enzyme inhibitor N-phenylthiourea. Our results underline the pivotal role of shell matrix in driving first CaCO3 deposition and the importance of tyrosinase in the formation of the first shell in M. galloprovincialis

Immunomodulation by Different Types of N-Oxides in the Hemocytes of the Marine Bivalve Mytilus galloprovincialis

The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO2, n-SiO2, n-ZnO, n-CeO2) chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may provide a basis for future experimental work for designing environmentally safer nanomaterials

Amphibian peptides for skin protection and healing

BACKGROUND: Amphibians are currently suffering a dramatic decline worldwide, mainly due to chytridiomycosis, a skin infection caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). An important natural defense of amphibian skin is the production of antimicrobial peptides (AMPs) by granular glands in the dermis. AMPs collected from several species of frogs successfully inhibit the growth of Bd in vitro. Besides their anti-microbial and anti-fungal activities, AMPs have been shown to exert other biological effects such as anti-viral, anti-tumor, anti-oxidant, immunomodulating and wound healing. AIM: We intended to test the efficacy of AMPs as cutaneous defenses in frog species either resistant or susceptible to Bd. METHODS: 3 frog species (Gastrotheca nebulanastes (GN), G. excubitor (GE) and Hypsiboas gladiator (HG), were collected in montane scrub, cloud forest and high elevation grassland habitats near Manu National Park in southeastern Peru. AMP secretion was stimulated by injection of norepinephrine into the dorsal lymph sacks. AMPs were then purified by chromatographic techniques. The human endothelial cell line HECV was treated with AMP concentrations ranging from 0.005 to 50 \ub5g/mL. Cell viability was verified by MTT test. Wound healing properties were analyzed by scratch wound assay. AMP inhibition strength against Bd growth was measured in vitro by incubating Bd zoospores with different concentrations of AMPs. RESULTS: Treatment with AMPs secreted from GN, GE and HG did not affect HECV cell viability at any concentration tested. No significant differences in cell migration rate were observed in HECV cells scratched and treated with GN and GE AMPs. Only HG peptides showed wound healing properties as well as strong Bd growth inhibiting ability. CONCLUSIONS: Stimulation of wound healing mechanisms and inhibition of Bd growth by skin AMPs might both contribute to HG resistance to chytridiomycosis. Understanding the role of skin defenses may lead to the development of novel Bd mitigation strategies. Possible applications of amphibian AMPs in skin medicine deserve attention and further studies. This work was funded by the European Commission (Tender ENV.B.3/SER/2016/0028, Mitigating a new infectious disease in salamanders to counteract the loss of European biodiversity) and by Parco Nazionale delle Cinque Terre

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

In vivo immunomodulatory and antioxidant properties of nanoceria (nCeO2) in the marine mussel Mytilus galloprovincialis

Cerium nanoparticles (nCeO2) are increasingly utilized in a wide variety of industrial, environmental and biomedical applications, and are therefore expected to be released in the aquatic environment. Due to its peculiar redox properties, nCeO2 may present unique hazards to environmental and human health. Previous data showed that in the hemocytes of the marine bivalve Mytilus galloprovincialis, in vitro exposure to a particular type of nCeO2 (9 nm, characterized by negative ζ-potential, high H2O2 scavenging capacity and Ce3+/Ce4+ surface ratio) reduced basal ROS production, lysosomal membrane stability and phagocytic activity in the presence of hemolymph serum; the effects observed were partly ascribed to the formation of a SOD-protein corona in the hemolymph. In this work, the in vivo effects of this type of nCeO2 were investigated in mussels exposed to 100 μg/L nCeO2 for 96 h; several lysosomal, immune, inflammatory and antioxidant biomarkers were measured at cellular (hemocytes) and tissue (gills, digestive gland) level. Molecular responses were evaluated in hemocytes and digestive gland by determining expression of 11 selected genes related to known biological functions. The results show specific immunomodulatory and antioxidant effects of nCeO2 at different levels of biological organization in the absence of Cerium tissue accumulation. These data further support the redox mechanisms at the basis of the physiological effects of nCeO2. Finally, in order to evaluate the possible impact at the whole organism level, the effects of nCeO2 were evaluated in the 48 h embryotoxicity assay in a wide concentration range. However, nCeO2 exposure resulted in a small reduction in normal embryo development. Overall, the results demonstrate that in mussels nCeO2 can selectively modulate different physiological processes at different levels of biological organization

Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32

none9Marine bivalves can accumulate large numbers of bacteria, in particular Vibrio species, whose persistence in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating hemocytes and hemolymph soluble factors. The interactions between vibrios and hemolymph have been investigated, in particular in bivalve species susceptible to infection by certain Vibrio spp. and strains. In this work, the effects of two bivalve pathogens, Vibrio splendidus LGP32 (V.s.) and Vibrio aestuarianus 01/032 (V.a.), isolated from oyster mortality outbreaks, on the hemocytes of Mytilus galloprovincialis were investigated. In vitro, V.s., but not V.a., induced a dramatic decrease in lysosomal membrane stability-LMS in the hemocytes; both vibrios induced a moderate lysozyme release, with V.s. > V.a.. The V.s.-induced decrease in LMS was mediated by activation of PI-3Kinase, as shown by use of different kinase inhibitors. TEM analysis showed rapid internalization of both vibrios; however, V.s. lead to cellular and lysosomal damage and was able to survive within the hemocytes, whereas significant killing of V.a. was observed. In vivo, in mussels challenged with either vibrio and sampled at 6, 24 and 96 h post-injection, transient decreases in hemocyte LMS and progressive increases in serum lysozyme activity were observed, with V.s. > V.a.. Moreover, whereas V.a. was efficiently cleared from hemolymph, V.s. showed significant growth, that was maximal at 24 h p.i. when lowest LMS values were recorded in the hemocytes. Both vibrios also induced significant decreases in LMS in the digestive gland, again with V.s. > V.a.. The results indicate distinct interactions between mussel hemocytes and the two vibrio strains tested. The effects of V.s. may be due to the capacity of this strain to interfere with the signaling pathways involved in hemocyte function, thus escaping the bactericidal activity of the host cell, as observed for certain mammalian pathogens. Although V.s. is considered not pathogenic to Mytilus, this vibrio strain can affect the lysosomal function at the cellular and tissue level, thus leading to stressful conditions.openBalbi, T; Fabbri, R; Cortese, K; Smerilli, A; Ciacci, C; Grande, C; Vezzulli, L; Pruzzo, C; Canesi, LBalbi, T; Fabbri, R; Cortese, K; Smerilli, A; Ciacci, C; Grande, C; Vezzulli, Luigi; Pruzzo, C; Canesi,

3,5-Diiodo-L-thyronine modulates the expression of genes of lipid metabolism in a rat model of fatty liver.

Recent reports demonstrated that 3,5-diiodo-l-thyronine (T(2)) was able to prevent lipid accumulation in the liver of rats fed a high-fat diet (HFD). In this study, we investigated how the rat liver responds to HFD and T(2) treatment by assessing the transcription profiles of some genes involved in the pathways of lipid metabolism: oxidation, storage and secretion. The mRNA levels of the peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARδ), and of their target enzymes acyl-CoA oxidase and stearoyl-CoA desaturase were evaluated by real-time RT-PCR. Moreover, the expression of the adipose triglyceride lipase involved in lipid mobilisation, of the main PAT proteins acting in lipid droplet (LD) turnover, and of apoprotein B (apo B), the major protein component of very low-density lipoproteins (VLDLs) were analysed. Overall, our data demonstrated that T(2) administration to HFD rats counteracts most of the hepatic transcriptional changes that occurred in response to the excess exogenous fat. In particular, our results suggest that T(2) may prevent the pathways leading to lipid storage in LDs, promote the processes of lipid mobilisation from LDs and secretion as VLDL, in addition to the stimulation of pathways of lipid oxidation. In conclusion, our findings might give an insight into the mechanisms underlying the anti-steatotic ability of T(2) and help to define the potential therapeutic role of T(2) for preventing or treating liver steatosis

The Organophosphate Chlorpyrifos Interferes with the Responses to 17β-Estradiol in the Digestive Gland of the Marine Mussel Mytilus galloprovincialis

BACKGROUND: Many pesticides have been shown to act as endocrine disrupters. Although the potencies of currently used pesticides as hormone agonists/antagonists are low compared with those of natural ligands, their ability to act via multiple mechanisms might enhance the biological effect. The organophosphate Chlorpyrifos (CHP) has been shown to be weakly estrogenic and cause adverse neurodevelopmental effects in mammals. However, no information is available on the endocrine effects of CHP in aquatic organisms. In the digestive gland of the bivalve Mytilus galloprovincialis, a target tissue of both estrogens and pesticides, the possible effects of CHP on the responses to the natural estrogen 17β-estradiol (E(2)) were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Mussels were exposed to CHP (4.5 mg/l, 72 hrs) and subsequently injected with E(2) (6.75 ng/g dw). Responses were evaluated in CHP, E(2) and CHP/E(2) treatment groups at 24 h p.i. by a biomarker/transcriptomic approach. CHP and E(2) induced additive, synergistic, and antagonistic effects on lysosomal biomarkers (lysosomal membrane stability, lysosome/cytoplasm volume ratio, lipofuscin and neutral lipid accumulation). Additive and synergistic effects were also observed on the expression of estrogen-responsive genes (GSTπ, catalase, 5-HTR) evaluated by RT-Q-PCR. The use of a 1.7K cDNA Mytilus microarray showed that CHP, E(2) and CHP/E(2), induced 81, 44, and 65 Differentially Expressed Genes (DEGs), respectively. 24 genes were exclusively shared between CHP and CHP/E(2), only 2 genes between E(2) and CHP/E(2). Moreover, 36 genes were uniquely modulated by CHP/E(2). Gene ontology annotation was used to elucidate the putative mechanisms involved in the responses elicited by different treatments. CONCLUSIONS: The results show complex interactions between CHP and E(2) in the digestive gland, indicating that the combination of certain pesticides and hormones may give rise to unexpected effects at the molecular/cellular level. Overall, these data demonstrate that CHP can interfere with the mussel responses to natural estrogens