Alessia Giannetto

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.

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.

Neurotoxicological effects on marine mussel Mytilus galloprovincialis caged at petrochemical contaminated areas (eastern Sicily, Italy): 1H NMR and immunohistochemical assays

The neurotoxicological potential of environmental pollution, mainly related to petrochemical activities, was investigated in marine mussel Mytilus galloprovincialis. Bivalve mollusks, particularly mussels, are widely used as sentinel organisms in biomonitoring studies for assessing the impact of anthropogenic contaminants. The gills, mainly involved in nutrient uptake, digestion, gas exchange and neuronal signaling, are the first organ to be affected by pollutants present in the external environment, and therefore were selected as the target organ for this study. Mussels from an aquaculture farm were caged at a highly polluted petrochemical area and a reference site along the Augusta coastline (eastern Sicily, Italy) for one month. A battery of biomarkers indicative of neuronal perturbations was applied on gills in order to investigate on the serotonergic (i.e. serotonin, 5-HT, and its receptor, 5-HT3R), cholinergic (i.e. acetylcholine, acetylcholinesterase, AChE, and choline acetyltransferase, ChAT), and dopaminergic systems (i.e. tyrosine and tyrosine hydroxylase, TH). Overall, impairment in the normal ciliary motility was found in mussels caged at the polluted site. Alterations in serotoninergic and cholinergic systems were revealed, with enhancement of dopaminergic neurotransmission resulting in a cilio-inhibitory effect. However, the over-expression in 5-HT3R and ChAT at cellular level may indicate an adaptive response of mussels to recover a regular physiological activity in gills. To our knowledge, this is the first study that uses (1)H NMR and immunohistochemical assays. Their concurrent use demonstrated to be sensitive and effective for assessing environmental influences on the health status of aquatic organisms, and thus suitable to be applied in ecotoxicological studies.

Ultrastructural and immunohistochemical investigation on the gills of the teleost, Thalassoma pavo L., exposed to cadmium

An investigation was conducted to determine the effects of the heavy metal, cadmium (Cd), on the gills of the teleost fish, Thalassoma pavo Linnaeus, 1758. The fishes were exposed to several sublethal concentrations of cadmium (10, 40, 60 and 120 μM (mg/L)) for a period of 48, 96 and 192 h. The value of the LC50 after 96 h of cadmium exposure, determined using the System of Finney, was equal to 128.3 μM. The gills of the fishes were examined by light and electron microscopy. Toxic, apoptotic and cadmium effects were analyzed using some neuropeptides, metallothioneins (MT), caspase 3, PCNA and calmodulin, as bioindicators, respectively. The results showed that the alterations in the gills were proportional to the exposure periods and concentrations of the metal, which were found to be both dose and time dependent. The biological responses in the gills of the tested animals are discussed in relation to results obtained by analysis of the biomarkers. These data may be used for the planning of a model to determine biological risk in the marine environment and may be particularly useful to investigate organisms exposed to cadmium.

A multibiomarker approach in Coris julis living in a natural environment.

To monitor the health of aquatic organisms, biomarkers have been used as effective tools in assessing environmental risk. In this study was examined the teleost Coris julis, sampled in two marine sites in Messina (Italy) at different pollution degree, Milazzo, characterized by a strong anthropogenic impact, and Marinello, the natural reserve. C. julis is a species particularly suitable to biomonitoring because its feeding habits favor bio-accumulation of xenobiotics. The following biomarkers were used to estimate the impact of highly persistent pollutants: cellular localization of cytochrome P4501A (CYP1A) and glutathione-S-transferase (GST) in the liver, their hepatic expression at the mRNA level, the enzymatic activity (EROD and BPMO), the micronucleus and comet assays in the blood, esterases (AChE in the brain and BChE in the blood) activity and evaluation of PAH metabolites in the bile. The present findings provide evidence of statistically significant differences in parameters between individuals collected in two sites.

Cellular biomarkers in the mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) from Lake Faro (Sicily, Italy)

Abstract Lake Faro (Sicily, Italy) is a natural confined brackish environment particularly subject to anthropogenic impact, resulting in a mixture of xenobiotic substances, i.e. heavy metals and polycyclic aromatic hydrocarbons (PAHs), and characterised by low hydrodynamics. In order to assess the water quality status of this pond, a multi-biomarker approach was applied on mussel Mytilus galloprovincialis (Lamarck, 1819) both inhabiting the lake and from a control site (Goro). Different biomarkers were investigated on mussel digestive glands and gills, including biomarkers of exposure (cytochrome P450 4, CYP4), neurotoxicity (acetylcholinesterase, AChE; choline acetyltransferase, ChAT), general stress (lysosomal membrane stability, LMS), and genotoxicity (micronucleus and comet assays). The results suggest significant responses related to the selected area. A statistically significant inhibition (P < 0.0001) of CYP4 in the digestive gland, and of AChE and ChAT in the gills, was found in specimens collected from Faro compared with the control. The lysosomal membrane stability of mussels from Lake Faro was lower than the reference site, while the DNA damages were significantly higher in mussels from the brackish area compared to the control. This study represents the first time the quality status of Lake Faro is assessed using a multi-biomarker approach on the mussel M. galloprovincialis, which proved to be suitable to identify the effects of environmental pollutants at molecular and cellular levels.

Daily Rhythmicity of Clock Gene Transcripts in Atlantic Cod Fast Skeletal Muscle

The classical notion of a centralized clock that governs circadian rhythmicity has been challenged with the discovery of peripheral oscillators that enable organisms to cope with daily changes in their environment. The present study aimed to identify the molecular clock components in Atlantic cod (Gadus morhua) and to investigate their daily gene expression in fast skeletal muscle. Atlantic cod clock genes were closely related to their orthologs in teleosts and tetrapods. Synteny was conserved to varying degrees in the majority of the 18 clock genes examined. In particular, aryl hydrocarbon receptor nuclear translocator-like 2 (arntl2), RAR-related orphan receptor A (rora) and timeless (tim) displayed high degrees of conservation. Expression profiling during the early ontogenesis revealed that some transcripts were maternally transferred, namely arntl2, cryptochrome 1b and 2 (cry1b and cry2), and period 2a and 2b (per2a and per2b). Most clock genes were ubiquitously expressed in various tissues, suggesting the possible existence of multiple peripheral clock systems in Atlantic cod. In particular, they were all detected in fast skeletal muscle, with the exception of neuronal PAS (Per-Arnt-Single-minded) domain-containing protein (npas1) and rora. Rhythmicity analysis revealed 8 clock genes with daily rhythmic expression, namely arntl2, circadian locomotor output cycles kaput (clock), npas2, cry2, cry3 per2a, nuclear receptor subfamily 1, group D, member 1 (nr1d1), and nr1d2a. Transcript levels of the myogenic genes myogenic factor 5 (myf5) and muscleblind-like 1 (mbnl1) strongly correlated with clock gene expression. This is the first study to unravel the molecular components of peripheral clocks in Atlantic cod. Taken together, our data suggest that the putative clock system in fast skeletal muscle of Atlantic cod has regulatory implications on muscle physiology, particularly in the expression of genes related to myogenesis.

Immunohistochemical and molecular biomarkers in Coris julis exposed to environmental contaminants

When a contaminant interacts with biotic components of a marine ecosystem, it causes a series of changes that can compromise an entire community (Stebbing, 1985). This present study wants to focus on changes in the gills of a bioindicator benthic organism, Coris julis, collected in Milazzo (Messina, Italy), characterized by a strong anthropical impact), compared with individuals from the control site (Marinello, Messina). RT-PCR has been used for both MT and HSP70, and the respective mRNAs have been visualized by FISH. MT and HSP70 expression levels increased in individuals collected in Milazzo. The presence of numerous apoptotic and proliferating cells and the analysis of several neuronal markers by immunohistochemical method give information about the adaptation to a heavy metal mixture. The obtained results show that, in stressed fishes, defensive processes increase to maintain the normal functions of the organs more exposed to the action of polluted substances.

Hypoxia-Inducible Factor α and Hif-prolyl Hydroxylase Characterization and Gene Expression in Short-Time Air-Exposed Mytilus galloprovincialis

Aquatic organisms experience environmental hypoxia as a result of eutrophication and naturally occurring tidal cycles. Mytilus galloprovincialis, being an anoxic/hypoxic-tolerant bivalve, provides an excellent model to investigate the molecular mechanisms regulating oxygen sensing. Across the animal kingdom, inadequacy in oxygen supply is signalled predominantly by hypoxia-inducible factors (HIF) and Hif-prolyl hydroxylases (PHD). In this study, hif-α 5′-end and partial phd mRNA sequences from M. galloprovincialis were obtained. Phylogenetic and molecular characterization of both HIF-α and PHD putative proteins showed shared key features with the respective orthologues from animals strongly suggesting their crucial involvement in the highly conserved oxygen sensing pathway. Both transcripts displayed a tissue-specific distribution with prominent expression in gills. Quantitative gene expression analysis of hif-α and phd mRNAs from gills of M. galloprovincialis demonstrated that both these key sensors are transcriptionally modulated by oxygen availability during the short-time air exposure and subsequent re-oxygenation treatments proving that they are critical players of oxygen-sensing mechanisms in mussels. Remarkably, hif-α gene expression showed a prompt and transient response suggesting the precocious implication of this transcription factor in the early phase of the adaptive response to hypoxia in Mytilus. HIF-α and PHD proteins were modulated in a time-dependent manner with trends comparable to mRNA expression patterns, thus suggesting a central role of their transcriptional regulation in the hypoxia tolerance strategies in marine bivalves. These results provide molecular information about the effects of oxygen deficiency and identify hypoxia-responsive biomarker genes in mussels applicable in ecotoxicological studies of natural marine areas.

Innervation and Neurotransmitter Localization in the Lung of the Nile bichir Polypterus bichir bichir

Anatomical and functional studies of the autonomic innervation in the lung of dipnoan fishes and the bichirs are lacking. The present immunohistochemical studies demonstrated the presence of nerve fibers in the muscle layers of the lung of the bichir, Polypterus bichir bichir, and identified the immunoreactive elements of this innervation. Tyrosine hydroxylase, acetylcholinesterase, and peptide immunoreactivity was detected in the intramural nerve fibers. Extensive innervation was present in the submucosa where adenylatecyclase/activating polypeptide 38, substance P, P2X2, and 5‐hydroxytryptamine (5‐HT)–immunoreactive nerve fibers mainly supplied blood vessels. A collection of monopolar neurons located in the submucosal and the muscular layers of the glottis expressed a variety of various transmitters. These neurons may be homologous to ganglion cells in the branchial and pharyngeal rami of the vagus in fishes. Nerves containing 5‐HT and P2X2 receptor immunoreactivity projected to the lung epithelium. Associated with neuroepithelial cells in mucociliated epithelium, were neuronal nitric oxide synthase–immunopositive axons. The physiological function of this innervation is not known. The present study shows that the pattern of autonomic innervation of the bichir lung may by similar in its elements to that in tetrapods. Anat Rec, 290:1166–1177, 2007.