Alessia Giannetto - Academia.edu (original) (raw)

Papers by Alessia Giannetto

Journal of Shellfish Research, 2016

Marine Environmental Research, 2016

This work was designed to evaluate the biological effects of petrochemical contamination on marin... more This work was designed to evaluate the biological effects of petrochemical contamination on marine mussels. Mytilus galloprovincialis, widely used as sentinel organisms in biomonitoring studies, were caged at the "Augusta-Melilli-Priolo" industrial site (eastern Sicily, Italy), chosen as one of the largest petrochemical areas in Europe, and Brucoli, chosen as reference site. Chemical analyses of sediments at the polluted site revealed high levels of PAHs and mercury, exceeding the national and international guideline limits. In mussels from the polluted site, severe morphological alterations were observed in gills, mainly involved in nutrient uptake and gas exchange. Changes in serotonergic and cholinergic systems, investigated through immunohistochemical, metabolomics and enzymatic approaches, were highlighted in gills, as well as onset of hypoxic adaptive responses with up-regulation of hypoxia-inducible factor transcript. Overall, the application of a multi-biomarker panel results effective in assessing the biological effects of petrochemical contamination on the health of aquatic organisms.

Marine Environmental Research, 2016

Persistent organic pollutants (POPs) are known to act as &amp... more Persistent organic pollutants (POPs) are known to act as "obesogens", being fat-soluble and affecting lipid metabolism. The Atlantic bluefin tuna, Thunnus thynnus, are top pelagic predators prone to bioaccumulate and biomagnify environmental contaminants. This study aimed at evaluating POPs-induced ectopic lipid accumulation in liver of adult tuna from the Mediterranean Sea. PCBs and organochlorine pesticides were measured in tuna liver, and marked morphological changes observed, namely poorly compacted tissues, intense vacuolization, erythrocyte infiltration and presence of melanomacrophages. The expression of perilipin, a lipid-droplet marker, positively correlated with the gene expression of PPARγ, a master regulator of adipogenesis, and its heterodimeric partner, RXRα. Changes in metabolites involved in fatty acid biosynthesis and ketogenesis were also observed. Although male bluefin tuna appeared to be more sensitive than females to the adverse effects of environmental obesogens, the alterations observed in tuna liver of both sexes suggest a potential onset of hepatic steatosis.

Marine biotechnology (New York, N.Y.), Jan 16, 2015

Aquatic organisms experience environmental hypoxia as a result of eutrophication and naturally oc... more 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 ...

Aquatic Toxicology, 2008

Metallothioneins (MTs), metal-inducible proteins, are crucial proteins for the regulation of esse... more 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 transcriptasepolymerase 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.

Terrestrial isopods respond quickly to heavy metals pollution and because they adopt a tolerance ... more Terrestrial isopods respond quickly to heavy metals pollution and because they adopt a tolerance strategy of immobilizing and accumulating heavy metals they act as excellent bioindicators of these pollutants. While in other crustaceans, especially decapods, the expression of MT has been well demonstrated, in terrestrial isopods, however, has only been suggested but never clearly verified (KNIGGE et al., 2002; ŽNIDARŠIČ et al., 2005). Our research, based on ICP-MS analysis, immunolocalization methods and western blotting, was aimed to evaluate, under laboratory conditions, the expression and localization of MT and Hsp70 in Armadillidium vulgare feeded with chestnut leaves contaminated with various sublethal concentrations of Cd and Pb. The Cd content of the animals treated with the various concentrations of the metal has been always higher than that of chestnut leaves contaminated. The uptake and accumulation of Pb was, instead, always modest compared to this of the chestnut leaves. ...

Lake Faro (Sicily, Italy) is a natural confined brackish environment particularly subject to anth... more 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.

Aquatic Toxicology, 2008

... higher concentration of Cd, Pb and Cr found in gills of M. galloprovincialis from Faro than s... more ... higher concentration of Cd, Pb and Cr found in gills of M. galloprovincialis from Faro than specimens from the reference site (Goro). ... These have been classified in two multicomponent gene families: MT-10 and MT-20 ( [Frazier, 1986] , [Mackay et al., 1993] , [Barsyte et al., 1999 ...

Marine Environmental Research, 2015

The embryotoxicity of CuO NPs was evaluated in the black sea urchin Arbacia lixula embryos, by us... more The embryotoxicity of CuO NPs was evaluated in the black sea urchin Arbacia lixula embryos, by using 24-well plates. Fertilized eggs were exposed to five doses of CuO NPs ranging from 0.07 to 20 ppb, until pluteus stage. CuO NPs suspensions in artificial seawater formed agglomerates of 80-200 nm size, and copper uptake was 2.5-fold up in larvae exposed to high NP concentrations in respect to control. Developmental delay and morphological alteration, including skeletal abnormalities, were observed, as well as impairment in cholinergic and serotonergic nervous systems. These findings suggest the potential of CuO NPs to interfere with the normal neurotransmission pathways, thus affecting larval morphogenesis. Overall, the embryotoxicity tests are effective for evaluation of nanoparticle effects on the health of aquatic biota. Furthermore, as the black sea urchin A. lixula demonstrated to be vulnerable to NP exposure, it may be a valid bioindicator in marine biomonitoring and ecotoxicological programmes.

Italian Journal of Zoology, 2009

The pituitary gland of swordfish (Xiphias gladius L.) was studied immunohistochemically using ant... more The pituitary gland of swordfish (Xiphias gladius L.) was studied immunohistochemically using antibodies against h-FSH b, h-LH b, GTH I b and GTH II b. The immunogold method for detection, at the ultrastructural level, of gonadotropins was also used. The immunostaining methods allowed identification of two distinct GTH I b (FSH-like) and GTH II b (LH-like) cells. Both GTH I b and GTH II b immunoreactive cells were observed in swordfish, although there were greater numbers of GTH II b immunoreactive cells. The localization and structure of the two different gonadotropic cells of swordfish are compared with other teleost arrangement. mRNA was isolated from X. gladius pituitaries and a series of oligonucleotides were constructed and used as primers in the polymerase chain reaction (PCR) to amplify GTH cDNAs. PCR products of eight independent PCR amplification reactions were cloned and sequenced for both GTH I b and GTH II b to avoid potential errors introduced during the PCR amplification. Two oligonucleotide probes were constructed and used in direct label fluorescence in situ hybridization (FISH) for measuring GTH I and GTH II mRNA expression.

Ecotoxicology and Environmental Safety, 2013

Multiple anthropogenic activities present along coastal environments may affect the health status... more Multiple anthropogenic activities present along coastal environments may affect the health status of aquatic ecosystems. In this study, specimens of European sea bass (Dicentrarchus labrax) were exposed for 30 days to highly contaminated sediment collected from the industrial area between Augusta and Priolo (Syracuse, Italy), defined as the most mercury polluted site in the Mediterranean. The aim was to evaluate the responses of juvenile D. labrax to highly contaminated sediments, particularly enriched in Hg, in order to enhance the scarce knowledge on the potential compensatory mechanisms developed by organisms under severe stress conditions. Apoptotic and proliferative activities [cell turnover: Proliferating Cell Nuclear Antigen (PCNA) and FAS Ligand (FasL)], onset of hypoxic condition [hypoxia: Hypoxia Inducibile Factor-1α (HIF-1α)], and changes in the neuroendocrine control mechanisms [neurotransmission: Tyrosine Hydroxylase (TH), Choline Acetyltransferase (ChAT), Acetylcholinesterase (AChE), 5-Hydroxytryptamine (5-HT) and 5-Hydroxytryptamine receptor 3 (5-HT3)] were investigated in sea bass gill tissues. In the specimens exposed to the polluted sediment, the occurrence of altered cell turnover may result in impaired gas exchange that leads to a condition of "functional hypoxia". Changes in neurotransmission pathways were also observed, suggesting a remodeling process as an adaptive response to increase the O 2 -carrying capacity and restore the normal physiological conditions of the gills. Overall, these findings demonstrated that although chronic exposure to heavy metal polluted sediments alters the functioning of both the nervous and endocrine systems, as well as plasticity of the gill epithelium, fish are able to trigger a series of physiological adjustments or adaptations interfering with specific neuroendocrine control mechanisms that enable their long-term survival.

Ecotoxicology and Environmental Safety, 2012

Environmental metabolomics was applied to assess the metabolic responses in transplanted mussels ... more 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 1 H 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.

PLoS ONE, 2012

Photoperiod is associated to phenotypic plasticity of somatic growth in several teleost species. ... more Photoperiod is associated to phenotypic plasticity of somatic growth in several teleost species. However, the molecular mechanisms underlying this phenomenon are currently unknown but it is likely that epigenetic regulation by methyltransferases is involved. The MLL (mixed-lineage leukaemia) family comprises histone methyltransferases that play a critical role in regulating gene expression during early development in mammals. So far, these genes have received scant attention in teleost fish. In the present study, the mean weight of Atlantic cod juveniles reared under continuous illumination was found to be 13% greater than those kept under natural photoperiod conditions for 120 days. We newly determined cDNA sequences of five mll (mll1, mll2, mll3a, mll4b and mll5) and two setd1 (setd1a and setd1ba) paralogues from Atlantic cod. Phylogenetic analysis revealed that the cod genes clustered within the appropriate mll clade and comparative mapping of mll paralogues showed that these genes lie within a region of conserved synteny among teleosts. All mll and setd1 genes were highly expressed in gonads and fast muscle of adult cod, albeit at different levels, and they were differentially regulated with photoperiod in muscle of juvenile fish. Following only one day of exposure to constant light, mll1, mll4b and setd1a were up to 57% lower in these fish compared to the natural photoperiod group. In addition, mRNA expression of myogenic regulatory factors (myog and myf-5) and pax7 in fast muscle was also affected by different photoperiod conditions. Notably, myog was significantly elevated in the continuous illumination group throughout the time course of the experiment. The absence of a day/night cycle is associated with a generalised decrease in mll expression concomitant with an increase in myog transcript levels in fast muscle of Atlantic cod, which may be involved in the observed epigenetic regulation of growth by photoperiod in this species.

Acta Histochemica, 2008

Immunohistochemical localisation of neurotransmitters was used to determine the distribution of u... more Immunohistochemical localisation of neurotransmitters was used to determine the distribution of unipolar neurons and neuroepithelial cells (NECs) in the respiratory tract of the bichir, Polypterus bichir bichir. NECs were commonly encountered in the mucociliated epithelium of the lung. Unipolar neurons were located in the submucosal and muscle layers of the glottis. The results suggest the presence of tyrosine hydroxylase (TH) and nNOS immunoreactivities in NECs. In addition, ACh-E/nNOS and TH/nNOS nerve fibers were also found associated with these cells. Unipolar neuronal cells showed a chemical code including the presence of 5-HT, ACh-E, peptides and P 2 Â 2 receptors. The present findings indicate that nitric oxide (NO) is a primitive transmitter of neuroepithelial oxygen-sensitive chemoreceptor cells together with acetylcholine. The coexistence of ACh-E with other substances in the unipolar neurons, but not with NO, may be a property of vagal postganglionic neurons since the emergence of the cranial autonomic pathways in the earliest vertebrates. It would be interesting to know about the provenance of the nerves in contact with NECs, which appear to have a complex innervation pattern.

PLoS ONE, 2014

The classical notion of a centralized clock that governs circadian rhythmicity has been challenge... more 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.

Developmental & Comparative Immunology, 2014

Continuous light treatment during early juvenile stages in Gadus morhua is a common farming manag... more Continuous light treatment during early juvenile stages in Gadus morhua is a common farming management practice but the effects of these unnatural light conditions on fish stress have received scant attention. In the present study we investigated how continuous illumination affects transcription levels of key stress-related and antimicrobial peptide genes in juvenile Atlantic cod. Gene expression quantification by real-time PCR revealed higher levels of transcripts coding for antioxidant enzymes, namely superoxide dismutase, catalase and glutathione reductase in liver of fish reared under continuous illumination, concomitantly with a 43% decrease in glutathione content. Transcription of antimicrobial peptides such as piscidins, hepcidin and cathelicidin was also affected by constant illumination. Overall, the significant changes in liver transcript levels of these biomarkers in response to continuous light may be an adaptation to light stress.

Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2015

The neurotoxicological potential of environmental pollution, mainly related to petrochemical acti... more 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-HT 3 R), 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-HT 3 R 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.

Gene, 2013

Photoperiod manipulation during early juvenile stages can influence growth in Atlantic cod. In th... more Photoperiod manipulation during early juvenile stages can influence growth in Atlantic cod. In the present study, one group of cod juveniles were reared under natural photoperiod conditions for Bodø (67°N, 14°E), whereas their counterparts were kept under continuous illumination. The mean weight of juvenile cod reared under continuous illumination was found to be 13% greater than those kept under natural photoperiod after 120 days of light treatment. The molecular basis of this phenotypic plasticity is currently unknown but it is likely that DNA (cytosine-5)-methyltransferases (dnmts) are involved, since these genes play a crucial role in epigenetic regulation of gene expression. Phylogenetic analysis of Atlantic cod dnmt1, dnmt2 and dnmt3a revealed that within each group, the phylogeny follows the taxonomic relationship between the various species and comparative mapping of dnmt paralogues showed that these genes lie within regions of conserved synteny amongst teleosts. Of the three dnmt paralogues, dnmt3a had the highest expression in fast muscle of adult cod. In addition, dnmt1 and dnmt2 were differentially expressed between tissues but with prominent expression in gonads. Dnmt1 and dnmt3a transcript levels showed a significant increase in fast muscle of juvenile cod from the continuous light group at several time points. Remarkably, dnmt1 and dnmt3a transcript levels were 2-fold higher at 120 days, by which point photoperiod conditions between the two light groups had become identical. Our data revealed that photoperiod can have an extended effect on expression of dnmt genes, which may be involved in the epigenetic regulation of muscle growth by photoperiod in Atlantic cod.

Acta Histochemica, 2011

An investigation was conducted to determine the effects of the heavy metal, cadmium (Cd), on the ... more 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 mM (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 mM. 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.

Journal of Shellfish Research, 2016

Marine Environmental Research, 2016

This work was designed to evaluate the biological effects of petrochemical contamination on marin... more This work was designed to evaluate the biological effects of petrochemical contamination on marine mussels. Mytilus galloprovincialis, widely used as sentinel organisms in biomonitoring studies, were caged at the &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;Augusta-Melilli-Priolo&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; industrial site (eastern Sicily, Italy), chosen as one of the largest petrochemical areas in Europe, and Brucoli, chosen as reference site. Chemical analyses of sediments at the polluted site revealed high levels of PAHs and mercury, exceeding the national and international guideline limits. In mussels from the polluted site, severe morphological alterations were observed in gills, mainly involved in nutrient uptake and gas exchange. Changes in serotonergic and cholinergic systems, investigated through immunohistochemical, metabolomics and enzymatic approaches, were highlighted in gills, as well as onset of hypoxic adaptive responses with up-regulation of hypoxia-inducible factor transcript. Overall, the application of a multi-biomarker panel results effective in assessing the biological effects of petrochemical contamination on the health of aquatic organisms.

Marine Environmental Research, 2016

Persistent organic pollutants (POPs) are known to act as &amp;amp;amp;amp;amp;amp;amp;amp;amp... more Persistent organic pollutants (POPs) are known to act as &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;obesogens&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;, being fat-soluble and affecting lipid metabolism. The Atlantic bluefin tuna, Thunnus thynnus, are top pelagic predators prone to bioaccumulate and biomagnify environmental contaminants. This study aimed at evaluating POPs-induced ectopic lipid accumulation in liver of adult tuna from the Mediterranean Sea. PCBs and organochlorine pesticides were measured in tuna liver, and marked morphological changes observed, namely poorly compacted tissues, intense vacuolization, erythrocyte infiltration and presence of melanomacrophages. The expression of perilipin, a lipid-droplet marker, positively correlated with the gene expression of PPARγ, a master regulator of adipogenesis, and its heterodimeric partner, RXRα. Changes in metabolites involved in fatty acid biosynthesis and ketogenesis were also observed. Although male bluefin tuna appeared to be more sensitive than females to the adverse effects of environmental obesogens, the alterations observed in tuna liver of both sexes suggest a potential onset of hepatic steatosis.

Marine biotechnology (New York, N.Y.), Jan 16, 2015

Aquatic organisms experience environmental hypoxia as a result of eutrophication and naturally oc... more 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 ...

Aquatic Toxicology, 2008

Metallothioneins (MTs), metal-inducible proteins, are crucial proteins for the regulation of esse... more 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 transcriptasepolymerase 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.

Terrestrial isopods respond quickly to heavy metals pollution and because they adopt a tolerance ... more Terrestrial isopods respond quickly to heavy metals pollution and because they adopt a tolerance strategy of immobilizing and accumulating heavy metals they act as excellent bioindicators of these pollutants. While in other crustaceans, especially decapods, the expression of MT has been well demonstrated, in terrestrial isopods, however, has only been suggested but never clearly verified (KNIGGE et al., 2002; ŽNIDARŠIČ et al., 2005). Our research, based on ICP-MS analysis, immunolocalization methods and western blotting, was aimed to evaluate, under laboratory conditions, the expression and localization of MT and Hsp70 in Armadillidium vulgare feeded with chestnut leaves contaminated with various sublethal concentrations of Cd and Pb. The Cd content of the animals treated with the various concentrations of the metal has been always higher than that of chestnut leaves contaminated. The uptake and accumulation of Pb was, instead, always modest compared to this of the chestnut leaves. ...

Lake Faro (Sicily, Italy) is a natural confined brackish environment particularly subject to anth... more 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.

Aquatic Toxicology, 2008

... higher concentration of Cd, Pb and Cr found in gills of M. galloprovincialis from Faro than s... more ... higher concentration of Cd, Pb and Cr found in gills of M. galloprovincialis from Faro than specimens from the reference site (Goro). ... These have been classified in two multicomponent gene families: MT-10 and MT-20 ( [Frazier, 1986] , [Mackay et al., 1993] , [Barsyte et al., 1999 ...

Marine Environmental Research, 2015

The embryotoxicity of CuO NPs was evaluated in the black sea urchin Arbacia lixula embryos, by us... more The embryotoxicity of CuO NPs was evaluated in the black sea urchin Arbacia lixula embryos, by using 24-well plates. Fertilized eggs were exposed to five doses of CuO NPs ranging from 0.07 to 20 ppb, until pluteus stage. CuO NPs suspensions in artificial seawater formed agglomerates of 80-200 nm size, and copper uptake was 2.5-fold up in larvae exposed to high NP concentrations in respect to control. Developmental delay and morphological alteration, including skeletal abnormalities, were observed, as well as impairment in cholinergic and serotonergic nervous systems. These findings suggest the potential of CuO NPs to interfere with the normal neurotransmission pathways, thus affecting larval morphogenesis. Overall, the embryotoxicity tests are effective for evaluation of nanoparticle effects on the health of aquatic biota. Furthermore, as the black sea urchin A. lixula demonstrated to be vulnerable to NP exposure, it may be a valid bioindicator in marine biomonitoring and ecotoxicological programmes.

Italian Journal of Zoology, 2009

The pituitary gland of swordfish (Xiphias gladius L.) was studied immunohistochemically using ant... more The pituitary gland of swordfish (Xiphias gladius L.) was studied immunohistochemically using antibodies against h-FSH b, h-LH b, GTH I b and GTH II b. The immunogold method for detection, at the ultrastructural level, of gonadotropins was also used. The immunostaining methods allowed identification of two distinct GTH I b (FSH-like) and GTH II b (LH-like) cells. Both GTH I b and GTH II b immunoreactive cells were observed in swordfish, although there were greater numbers of GTH II b immunoreactive cells. The localization and structure of the two different gonadotropic cells of swordfish are compared with other teleost arrangement. mRNA was isolated from X. gladius pituitaries and a series of oligonucleotides were constructed and used as primers in the polymerase chain reaction (PCR) to amplify GTH cDNAs. PCR products of eight independent PCR amplification reactions were cloned and sequenced for both GTH I b and GTH II b to avoid potential errors introduced during the PCR amplification. Two oligonucleotide probes were constructed and used in direct label fluorescence in situ hybridization (FISH) for measuring GTH I and GTH II mRNA expression.

Ecotoxicology and Environmental Safety, 2013

Multiple anthropogenic activities present along coastal environments may affect the health status... more Multiple anthropogenic activities present along coastal environments may affect the health status of aquatic ecosystems. In this study, specimens of European sea bass (Dicentrarchus labrax) were exposed for 30 days to highly contaminated sediment collected from the industrial area between Augusta and Priolo (Syracuse, Italy), defined as the most mercury polluted site in the Mediterranean. The aim was to evaluate the responses of juvenile D. labrax to highly contaminated sediments, particularly enriched in Hg, in order to enhance the scarce knowledge on the potential compensatory mechanisms developed by organisms under severe stress conditions. Apoptotic and proliferative activities [cell turnover: Proliferating Cell Nuclear Antigen (PCNA) and FAS Ligand (FasL)], onset of hypoxic condition [hypoxia: Hypoxia Inducibile Factor-1α (HIF-1α)], and changes in the neuroendocrine control mechanisms [neurotransmission: Tyrosine Hydroxylase (TH), Choline Acetyltransferase (ChAT), Acetylcholinesterase (AChE), 5-Hydroxytryptamine (5-HT) and 5-Hydroxytryptamine receptor 3 (5-HT3)] were investigated in sea bass gill tissues. In the specimens exposed to the polluted sediment, the occurrence of altered cell turnover may result in impaired gas exchange that leads to a condition of "functional hypoxia". Changes in neurotransmission pathways were also observed, suggesting a remodeling process as an adaptive response to increase the O 2 -carrying capacity and restore the normal physiological conditions of the gills. Overall, these findings demonstrated that although chronic exposure to heavy metal polluted sediments alters the functioning of both the nervous and endocrine systems, as well as plasticity of the gill epithelium, fish are able to trigger a series of physiological adjustments or adaptations interfering with specific neuroendocrine control mechanisms that enable their long-term survival.

Ecotoxicology and Environmental Safety, 2012

Environmental metabolomics was applied to assess the metabolic responses in transplanted mussels ... more 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 1 H 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.

PLoS ONE, 2012

Photoperiod is associated to phenotypic plasticity of somatic growth in several teleost species. ... more Photoperiod is associated to phenotypic plasticity of somatic growth in several teleost species. However, the molecular mechanisms underlying this phenomenon are currently unknown but it is likely that epigenetic regulation by methyltransferases is involved. The MLL (mixed-lineage leukaemia) family comprises histone methyltransferases that play a critical role in regulating gene expression during early development in mammals. So far, these genes have received scant attention in teleost fish. In the present study, the mean weight of Atlantic cod juveniles reared under continuous illumination was found to be 13% greater than those kept under natural photoperiod conditions for 120 days. We newly determined cDNA sequences of five mll (mll1, mll2, mll3a, mll4b and mll5) and two setd1 (setd1a and setd1ba) paralogues from Atlantic cod. Phylogenetic analysis revealed that the cod genes clustered within the appropriate mll clade and comparative mapping of mll paralogues showed that these genes lie within a region of conserved synteny among teleosts. All mll and setd1 genes were highly expressed in gonads and fast muscle of adult cod, albeit at different levels, and they were differentially regulated with photoperiod in muscle of juvenile fish. Following only one day of exposure to constant light, mll1, mll4b and setd1a were up to 57% lower in these fish compared to the natural photoperiod group. In addition, mRNA expression of myogenic regulatory factors (myog and myf-5) and pax7 in fast muscle was also affected by different photoperiod conditions. Notably, myog was significantly elevated in the continuous illumination group throughout the time course of the experiment. The absence of a day/night cycle is associated with a generalised decrease in mll expression concomitant with an increase in myog transcript levels in fast muscle of Atlantic cod, which may be involved in the observed epigenetic regulation of growth by photoperiod in this species.

Acta Histochemica, 2008

Immunohistochemical localisation of neurotransmitters was used to determine the distribution of u... more Immunohistochemical localisation of neurotransmitters was used to determine the distribution of unipolar neurons and neuroepithelial cells (NECs) in the respiratory tract of the bichir, Polypterus bichir bichir. NECs were commonly encountered in the mucociliated epithelium of the lung. Unipolar neurons were located in the submucosal and muscle layers of the glottis. The results suggest the presence of tyrosine hydroxylase (TH) and nNOS immunoreactivities in NECs. In addition, ACh-E/nNOS and TH/nNOS nerve fibers were also found associated with these cells. Unipolar neuronal cells showed a chemical code including the presence of 5-HT, ACh-E, peptides and P 2 Â 2 receptors. The present findings indicate that nitric oxide (NO) is a primitive transmitter of neuroepithelial oxygen-sensitive chemoreceptor cells together with acetylcholine. The coexistence of ACh-E with other substances in the unipolar neurons, but not with NO, may be a property of vagal postganglionic neurons since the emergence of the cranial autonomic pathways in the earliest vertebrates. It would be interesting to know about the provenance of the nerves in contact with NECs, which appear to have a complex innervation pattern.

PLoS ONE, 2014

The classical notion of a centralized clock that governs circadian rhythmicity has been challenge... more 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.

Developmental & Comparative Immunology, 2014

Continuous light treatment during early juvenile stages in Gadus morhua is a common farming manag... more Continuous light treatment during early juvenile stages in Gadus morhua is a common farming management practice but the effects of these unnatural light conditions on fish stress have received scant attention. In the present study we investigated how continuous illumination affects transcription levels of key stress-related and antimicrobial peptide genes in juvenile Atlantic cod. Gene expression quantification by real-time PCR revealed higher levels of transcripts coding for antioxidant enzymes, namely superoxide dismutase, catalase and glutathione reductase in liver of fish reared under continuous illumination, concomitantly with a 43% decrease in glutathione content. Transcription of antimicrobial peptides such as piscidins, hepcidin and cathelicidin was also affected by constant illumination. Overall, the significant changes in liver transcript levels of these biomarkers in response to continuous light may be an adaptation to light stress.

Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2015

The neurotoxicological potential of environmental pollution, mainly related to petrochemical acti... more 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-HT 3 R), 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-HT 3 R 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.

Gene, 2013

Photoperiod manipulation during early juvenile stages can influence growth in Atlantic cod. In th... more Photoperiod manipulation during early juvenile stages can influence growth in Atlantic cod. In the present study, one group of cod juveniles were reared under natural photoperiod conditions for Bodø (67°N, 14°E), whereas their counterparts were kept under continuous illumination. The mean weight of juvenile cod reared under continuous illumination was found to be 13% greater than those kept under natural photoperiod after 120 days of light treatment. The molecular basis of this phenotypic plasticity is currently unknown but it is likely that DNA (cytosine-5)-methyltransferases (dnmts) are involved, since these genes play a crucial role in epigenetic regulation of gene expression. Phylogenetic analysis of Atlantic cod dnmt1, dnmt2 and dnmt3a revealed that within each group, the phylogeny follows the taxonomic relationship between the various species and comparative mapping of dnmt paralogues showed that these genes lie within regions of conserved synteny amongst teleosts. Of the three dnmt paralogues, dnmt3a had the highest expression in fast muscle of adult cod. In addition, dnmt1 and dnmt2 were differentially expressed between tissues but with prominent expression in gonads. Dnmt1 and dnmt3a transcript levels showed a significant increase in fast muscle of juvenile cod from the continuous light group at several time points. Remarkably, dnmt1 and dnmt3a transcript levels were 2-fold higher at 120 days, by which point photoperiod conditions between the two light groups had become identical. Our data revealed that photoperiod can have an extended effect on expression of dnmt genes, which may be involved in the epigenetic regulation of muscle growth by photoperiod in Atlantic cod.

Acta Histochemica, 2011

An investigation was conducted to determine the effects of the heavy metal, cadmium (Cd), on the ... more 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 mM (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 mM. 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.