Salvatore Fasulo

Metabolomic investigation of Mytilus galloprovincialis (Lamarck 1819) caged in aquatic environments

Environmental metabolomics was applied to assess the metabolic responses in transplanted mussels to environmental pollution. Specimens of Mytilus galloprovincialis, sedentary filter-feeders, were caged in anthropogenic-impacted and reference sites along the Augusta coastline (Sicily, Italy). Chemical analysis revealed increased levels of PAHs in the digestive gland of mussels from the industrial area compared with control, and marked morphological changes were also observed. Digestive gland metabolic profiles, obtained by 1H NMR spectroscopy and analyzed by multivariate statistics, showed changes in metabolites involved in energy metabolism. Specifically, changes in lactate and acetoacetate could indicate increased anaerobic fermentation and alteration in lipid metabolism, respectively, suggesting that the mussels transplanted to the contaminated field site were suffering from adverse environmental condition. The NMR-based environmental metabolomics applied in this study results thus in it being a useful and effective tool for assessing environmental influences on the health status of aquatic organisms.

Effects of environmental pollution in caged mussels (Mytilus galloprovincialis)

Biological effects of environmental pollution, mainly related to presence of PAHs, were assessed in mussels Mytilus galloprovincialis caged in Priolo, an anthropogenically-impacted area, and Vendicari, a reference site, both located along the eastern coastline of Sicily (Italy). PAHs concentration and histopathological changes were measured in digestive gland tissues. Expression of cytochrome P4504Y1 (CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and activity of catalase (CAT) as oxidative stress index, were evaluated. The results show a direct correlation between the high concentrations of PAHs in digestive glands of mussels from Priolo and the significantly altered activity of phase I (P < 0.001) and phase II (P < 0.0001) biotransformation enzymes, along with increased levels of CAT activity (P < 0.05). These findings show the enhancement of the detoxification and antioxidant defense systems. The mussel caging approach and selected biomarkers demonstrated to be reliable for the assessment of environmental pollution effects on aquatic organisms.

Enhanced toxicity of 'bulk' titanium dioxide compared to 'fresh' and 'aged' nano-TiO2 in marine mussels (Mytilus galloprovincialis).

Abstract Marine bivalves (Mytilus galloprovincialis) were exposed to titanium dioxide (10 mg L−1) either as engineered nanoparticles (nTiO2; fresh, or aged under simulated sunlight for 7 days) or the bulk equivalent. Inductively coupled plasma-optical emission spectrometry analyses of mussel tissues showed higher Ti accumulation (>10-fold) in the digestive gland compared to gills. Nano-sized TiO2 showed greater accumulation than bulk, irrespective of ageing, particularly in digestive gland (>sixfold higher). Despite this, transcriptional expression of metallothionein genes, histology and histochemical analysis suggested that the bulk material was more toxic. Haemocytes showed significantly enhanced DNA damage, determined by the modified comet assay, for all treatments compared to the control, but no significant differences between the treatments. Our integrated study suggests that for this ecologically relevant organism photocatalytic ageing of nTiO2 does not significantly alter toxicity, and that bulk TiO2 may be less ecotoxicologically inert than previously assumed.

Impact of environmental pollution on caged mussels Mytilus galloprovincialis using NMR-based metabolomics

Metabolic responses to environmental pollution, mainly related to Hg and PAHs, were investigated in mussels. Specimens of Mytilus galloprovincialis, sedentary filter-feeders, were caged in anthropogenic-impacted and reference sites along the Augusta coastline (Sicily, Italy). The gills, mainly involved in nutrient uptake, digestion and gas exchange, were selected as target organ being the first organ to be affected by pollutants. Severe alterations in gill tissue were observed in mussels from the industrial area compared with control, while gill metabolic profiles, obtained by (1)H NMR spectroscopy and analyzed by multivariate statistics, exhibited significant changes in amino acids, energy metabolites, osmolytes and neurotransmitters. Overall, the morphological changes and metabolic disturbance detected in gill tissues may suggest that the mussels transplanted to the contaminated field site were suffering from adverse environmental condition. The concurrent morphological and metabolomic investigations as applied here result effective in assessing the environmental influences on health status of aquatic organisms.

Effects of sublethal, environmentally relevant concentrations of hexavalent chromium in the gills of Mytilus galloprovincialis.

Hexavalent chromium Cr(VI) is an important contaminant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. In the marine bivalve Mytilus galloprovincialis exposure to non-toxic, environmentally relevant concentrations of Cr(VI) was shown to modulate functional parameters and gene expression in both the digestive gland and hemocytes. In this work, the effects of exposure to Cr(VI) (0.1-1-10 μg L(-1) animal(-1) for 96 h) in mussel gills were investigated. Gill morphology and immunolocalization of GSH-transferase (GST), of components involved in cholinergic (AChE and ChAT), adrenergic (TH) and serotoninergic (5-HT(3) receptor) systems, regulating gill motility, were evaluated. Total glutathione content, activities of GSH-related enzymes (glutathione reductase - GSR, GST), of catalase, and of key glycolytic enzymes (phosphofructokinase - PFK and pyruvate kinase - PK) were determined. Moreover, mRNA expression of selected Mytilus genes (GST-π, metallothionein isoforms MT10 and MT20, HSP70 and 5-HT receptor) was assessed by RT-q-PCR. Cr(VI) exposure induced progressive changes in gill morphology and in immunoreactivity to components involved in neurotransmission that were particularly evident at the highest concentration tested, and associated with large metal accumulation. Cr(VI) increased the activities of GST and GSR, and total glutathione content to a different extent at different metal concentrations, this suggesting Cr(VI) detoxication/reduction at the site of metal entry. Cr(VI) exposure also increased the activity of glycolytic enzymes, indicating modulation of carbohydrate metabolism. Significant changes in transcription of different genes were observed. In particular, the mRNA level for the 5-HTR was increased, whereas both decreases and increases were observed for GST-π, MT10, MT20 and HSP70 mRNAs, showing sex- and concentration-related differences. The results demonstrate that Cr(VI) significantly affected functional and molecular parameters in mussel gills, and indicate that this tissue represents the major target of exposure to environmentally relevant concentrations of the metal.

Structural, histochemical and functional aspects of the epidermis of fishes

Significant progress has been made in the last 30 years in our knowledge of cellular and physiological aspects of fish epidermis This review surveys the histology, histochemistry (including lectin- and immunohistochemistry) and function of the epidermis of various teleosts and cyclostomes in the light of the new data. The epidermis is a multipurpose tissue, although the secretory function is dominant. Mucous secretions are produced by different types of epithelial cells, in particular those located in the superficial layer. Specific unicellular glands in the skin of gnathostome fish are the goblet cells, the sacciform cells and the club cells. In addition, lampreys and hagfish possess specific secretory cell types. Other specialized types are the holocrine venom cells which are aggregated in venom glands, the mitochondria-rich cells (ionocytes) and the photocytes, the latter grouped with other cell types to form photophores that are embedded in the skin. The outer epidermal cells and the mucous goblet cells secrete the epithelial mucous layer, whose carbohydrate composition changes with stress and environmental conditions. The mucus contains neuronal and endothelial forms of nitric oxide synthase (NOS), the enzyme responsible for the production of nitric oxide (NO), a prominent vascular and neuronal messenger that regulates many epithelial functions. The cytokeratins in the skin tissues have different distribution patterns in the various cell types, correlated with specific epithelial differentiation or, in hagfish, the modulation of the viscoelastic properties of skin mucus Club cell and sacciform cell secretions are now considered as storehouses of biologically active substances. Their expression is paralleled with that present in glandular cells of the skin of other lower vertebrates. The fine structure and histochemical properties of club cells are clearly different from those of the venom cells in fish and amphibians. Mitochondria-rich chloride cells (ionocytes), although primarily concentrated in the branchial epithelium, also occur in various areas of the skin. The skin chloride cells, like those in the gill, secrete monovalent ions in sea water and take up ions in fresh water. Fish skin has a diffuse paraneuronal cell system responsible for the production of bioactive compounds that have possible regulating functions. Neuropeptides occur in the Merkel cells, the mechanoreceptors (neuromast hair cells) and the electroreceptors (sensory cells of the ampullary and tuberous organs) of the skin. Basal cells of the taste buds are the source of neuron-specific enolase, serotonin, bombesin and somatostatin. Photophores contain serotonin and adrenalin, which play a neuromodulatory role in the regulation of luminescence. The functions of the skin secretions are primarily protective, as a result of their antimicrobial properties. Further analysis of the components of epidermal cells and unicellular glands of aquatic animals will disclose many protective factors

Distribution patterns of the paraneuronal endocrine cells in the skin, gills and the airways of fishes as determined by immunohistochemical and histological methods

SummaryThe neuro-endocrine cells of fish skin and respiratory surfaces, and their bioactive secretion as far as is known, are reviewed, and compared with similar elements in tetrapods, particularly amphibians. In the skin of teleost fish, immunohistochemistry has shown that Merkel cells react for serotonin, neuron-specific enolase and enkephalins. The pharmacology is not established in dipnoans or lampreys. In some teleosts, neuromasts react for substance P and leu-enkephalins; substance P is also reported from some ampullary organs (electroreceptors). Taste buds of teleosts may react for enkephalin and substance P. Basal cells of taste buds react for serotonin and neuron-specific enolase. Some unicellular skin glands of teleosts express bioactive compounds, including serotonin and some peptides; this ectopic expression is paralleled in amphibian skin glands. The dipnoan Protopterus has innervated pulmonary neuro-endocrine cells in the pneumatic duct region with dense-cored vesicles. In Polypterus and Amia the lungs have serotonin-positive neuro-endocrine cells that are apparently not innervated. In fish gills, a closed type of neuro-endocrine cell reacts for serotonin, an open type for enkephalins and some calcium-binding proteins (calbindin, calmodulin and S-100 protein). The functions of neuro-endocrine cells in fishes await investigation, but it is assumed they are regulatory.

Expression of metallothionein mRNAs by in situ hybridization in the gills of Mytilus galloprovincialis, from natural polluted environments

Metallothioneins (MTs), metal-inducible proteins, are crucial proteins for the regulation of essential metals, and are transcriptionally induced in all organisms by certain heavy metals, oxidative stress and inflammation. The gills represent an organ of uptake and loss of metals in which different mechanisms are present controlling the functions directly involved in the maintenance of homeostasis. In this study, the morphological and histomorphological aspects of branchial epithelium in Mytilus galloprovincialis from polluted environment (Faro swamp, Messina, Italy) have been investigated. The reverse transcriptase-polymerase chain reaction (PCR) has been used to isolate complementary DNA of both MT isoforms present from RNA extracted from mussel gills. The respective mRNAs on histological sections have been visualized by in situ hybridization. These methods showed that MT-10 mRNA is expressed at the basal level. In contrast, the MT-20 expression level was very low under basal conditions, while its mRNA increased dramatically in individuals collected in Faro. The presence of acid mucocytes and MTs in the gills may be considered a further defensive mechanism also related to the significantly higher concentration of Cd, Pb and Cr found in gills of M. galloprovincialis from Faro than specimens from the reference site (Goro). The results obtained show that, in stressed mussels, the defensive processes increase to maintain the normal functions of the organs more exposed to the action of polluted substances.

NANC nerves in the respiratory air sac and branchial vasculature of the indian catfish, Heteropneustes fossilis*

Gill and air sac of the Indian catfish Heteropneustes fossilis harbour a nerve network comprising an innervated system of neuroepithelial endocrine cells; the latter cells are found especially in the gill. A series of antibodies was used for the immunohistochemical detection of neurotransmitters of the neural non-adrenergic, non-cholinergic (NANC) systems such as the sensory neuropeptides (enkephalins), the inhibitory neuropeptide VIP and neuronal nitric oxide synthase (nNOS) responsible for nitric oxide (NO) production which is an inhibitory NANC neurotransmitter. NADPH-diaphorase (NADPH-d) histochemistry was used as marker of nNOS although it is not a specific indicator of constitutively-expressed NOS in gill and air sac tissues. A tyrosine hydroxylase antibody was used to investigate adrenergic innervation. Nitrergic and VIP-positive sensory innervation was found to be shared by gill and air sac. Immunohistochemistry revealed the presence of enkephalins, VIP, NOS and NADPH-d in nerves associated with branchial and air sac vasculature, and in the neuroendocrine cell systems of the gill. Adrenergic nerve fibers were found in some parts of the air sac vasculature. The origin of the nerve fibers remains unclear despite previous findings showing the presence of both NADPH-d and nNOS in the sensory system of the glossopharyngeal and vagus nerves including the branchial structure. Scarce faintly stained nNOS-positive neurons were located in the gill but were never detected in the air sac. These findings lead to the conclusion that a postganglionic innervation of the airways is absent. Mucous goblet cells in the gill were found to express nNOS and those located in the non-respiratory interlamellar areas of the air sac were densely innervated by nNOS-positive and VIP-positive nerve fibers. Our immunohistochemical studies demonstrate that most arteries of the gill and air sac share a NANC (basically nitrergic) innervation which strongly suggests that they are homologous structures.

Neuronal nitric oxide synthase (nNOS) expression in the epithelial neuroendocrine cell system and nerve fibers in the gill of the catfish, Heteropneustes fossilis.

We studied immunohistochemically the localization of neuronal nitric oxide synthase (nNOS) in gills of an Indian catfish species, Heteropneustes fossilis. It is shown that most of the epithelial neuroendocrine cells that are present in gill filaments and lamellae stained positively. Co-localization of nNOS and endothelin was also shown in neuroendocrine cells. A dense plexus of nNOS-containing nerve fibers was present beneath the gill epithelium, associated with efferent filament arteries and the basal side of neuroendocrine cells. nNOS immunopositive neurons were not found in gill areas. nNOS immunopositive neuroendocrine cells appeared to differ from neuroepithelial cells in gills of various teleost species, which are considered as oxygen-sensitive receptors and are present in the distal halves of gill filaments. Other types of neuroendocrine cells have been identified previously in other areas of gills using antibodies to serotonin and endothelin peptides. These cell types are likely to be involved in chemical regulation of the physiology of gill cells. In relation to the function of the other cell types, our data on nNOS localization suggest that NO is a wide-spread transmitter in the gill of the Indian catfish. It may play a role both in the local regulation of vascular tone and in inhibitory innervation of the gill.