Antonino Natalotto

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.

Effects of "in vivo" exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax).

Aquatic ecosystems are affected by all the impacts generated by a variety of anthropogenic activities present along coastal environments. The sediment compartment is the final receptor of water-insoluble pollutants, acting both as a sink and as a source of pollutants to the water column, and affecting both nektonic and benthic organisms. The aim of this study is to assess the impact of metals in the sediments collected from two sites in the petrochemical area between Augusta and Priolo (SR, Sicily, Italy) on gills of Dicentrarchus labrax. This was done to enhance the scarce knowledge on the bioavailability of metals bound to sediment and their capacity to interact with the bioindicator species. Various sublethal endpoints were assessed such as histopathological lesions, metallothioneins (MTs) and molecules involved in the homeostasis pathways by immunolocalization and RT-PCR. In the specimens exposed to sediments, the data suggested a reduction of gill cell membrane permeability, which could result in altered osmotic balance and gas exchange. Further, an increase of MT expression was detected, consisted the involvement of this protein in detoxification of toxic non-essential metals. The findings of this study demonstrate that a subchronic test, conducted by using sensitive and sub-lethal endpoints, in combination with chemical analyses, is a powerful tool for early identification of environmental hazards associated with contaminated sediments.

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.

Haemolytic activity and characterization of nematocyst venom from Pelagia noctiluca (Cnidaria: Scyphozoa)

Abstract We investigated the haemolytic capacity of the crude venom extracted from isolated nematocysts of Pelagia noctiluca (Cnidaria: Scyphozoa), and evidenced the proteic fractions responsible for this activity. The nematocyst venom was used at various concentrations to evaluate the haemolytic activity and the lysosomal membrane stability of red blood cells of two teleostean species treated with the extract. The nematocyst extract was assayed against erythrocytes of the two teleostean species living in different environments, Carassius auratus as a common freshwater species, and Liza aurata as a representative of seawater species. Experiments on the haemolytic activity of P. noctiluca in the presence of lipid components of erythrocyte membranes showed that sphyngomyelin strongly inhibited this activity. The crude venom was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE and high performance liquid chromatography (HPLC) to detect the proteic composition, and it was found that the active haemolytic components of this venom are distributed in at least four protein fractions. The results of our experiments indicated that Pelagia noctiluca venom induces haemolysis and lysosomal membrane destabilisation in both species and that Carassius auratus was more susceptible to jellyfish venom than was Liza aurata. No significant differences in glutathione (GSH) levels were observed between control and treatments; consequently the toxins do not cause the oxidative stress but likely recognize specific targets (i.e. sphyngomyelin) in the plasmatic membrane of red blood cells.

Biomarkers of environmental stress in gills of Pinna nobilis (Linnaeus 1758) from Balearic Island.

UNLABELLED In aquatic environments, bivalve molluscs are used as sentinel species for environmental biomonitoring. In this study Pinna nobilis specimens, the biggest Mediterranean bivalve, were collected in the Magaluf bay (Mallorca), a touristic location and in a pristine area of the Cabrera National Park as the control location. Histological and histochemical analysis in gills of specimens sampled from Magaluf exhibited evident tissue alterations with high presence of haemocytes. Lower acetylcholinesterase (AChE) activity and protein expression were also found in the gills of specimens collected from Magaluf compared with the control area. The determination of antioxidant enzyme activities, such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, showed a higher activities of these antioxidant enzymes and total glutathione content in samples from Magaluf bay than in Cabrera. In conclusion, the present study demonstrated that human activities result in morphological tissue alterations and a reduced AChE activity in gills of P. nobilis. Moreover, these stressful environmental conditions induced an adaptive response in P. nobilis as evidenced by increased antioxidant defences and a decreased AChE activity. CAPSULE The human activities induce oxidative stress in P. nobilis as evidenced by increased antioxidant defences and a decreased acetylcholinesterase activity.

Increased antioxidant response and capability to produce ROS in hemocytes of Pinna nobilis L. exposed to anthropogenic activity

Environmental pollutants exert immunotoxical effects on aquatic organisms. The aim was to determine the antioxidant response, markers of oxidative damage and reactive oxygen species production in hemocytes of Pinna nobilis, the largest endemic bivalve in the Mediterranean Sea, under anthropogenic pressure. P. nobilis individuals were collected from two locations along Mallorca Island waters attending to different degree of human impact and the hemocytes were obtained. Specimens from the impacted area showed increased activities of the antioxidant enzymes - catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase -, myeloperoxidase activity and reduced glutathione levels. No differences in oxidative damage markers - malondiahdehyde and carbonyl index - were evidenced between the pristine and polluted areas. Hemocytes from the polluted area presented increased capability to generate reactive oxygen species and nitrite/nitrate when activated. In conclusion, the human activities primed hemocytes for oxidative burst and increased the antioxidant mechanism without evidence of oxidative damage.

INFLUENCES OF ENVIRONMENTAL VARIABLES ON NEUROTRANSMISSION, OXIDATIVE SYSTEM, AND HYPOXIA SIGNALING ON TWO CLAM SPECIES FROM A MEDITERRANEAN COASTAL LAGOON

ABSTRACT Capo Peloro Lagoon is a coastal brackish system in northeastern Sicily (Italy), consisting of Ganzirri Lake and Faro Lake. The biological effects related to physicochemical parameters were assessed in two autochthonous clam species, Tapes decussatus and Tapes laeta, chosen for their abundance and economic interest, collected from the two Ganzirri Lake subbasins. Higher temperature and salinity values along with lower dissolved oxygen levels were recorded at the southernmost (site B) relatively to northernmost (site A) subbasin. A battery of biomarkers was investigated in clam gills, including histomorphological endpoint, cholinergic neurotransmission, oxidative stress, and hypoxia biomarkers. Both clam species from site B showed altered gill histomorphology attributable to inflammatory processes. A compromised cholinergic neurotransmission, stimulation of the antioxidant defense system, and onset of hypoxic adaptive responseswere also observed. Overall, data suggest that the environmental variables, as those recorded in the southernmost subbasin ofGanzirri Lake, adversely interfere with the physiological function of the gills, triggering a number of adaptive responses in the clams.

Effects of Oxygen Availability on Oxidative Stress Biomarkers in the Mediterranean Mussel Mytilus galloprovincialis

In aquatic environments, hypoxia and oxygen-deficient areas are increasing worldwide. Transitions in oxygen levels can influence the production of reactive oxygen species (ROS), eventually leading to oxidative stress. The transcriptional response of oxidative stress biomarkers was evaluated by qPCR in gill tissue from Mytilus galloprovincialis experimentally subjected to 48-h air exposure followed by 48-h re-oxygenation, as compared to normoxic control mussels. Superoxide dismutases (CuZnsod and Mnsod), catalase (cat), and glutathione S-transferase (gst) were over-expressed early after 8-h air exposure and returned to normoxic levels during re-oxygenation. Moreover, the mRNAs and protein expression patterns of heat shock proteins (HSP70 and HSP90) and metallothioneins (MT-10 and MT-20) were modulated by oxygen availability with increased levels during re-oxygenation suggesting the participation of these cytoprotective mechanisms in the physiological oxidative stress response when oxygen concentration was restored. Overall, the observed modulation of the oxidative stress-related and general stress genes indicates that M. galloprovincialis responds to changes in oxygen availability enhancing the antioxidant potential under low oxygen conditions for dealing with the oxidative burst during future re-oxygenation. The present investigation brings further insights in understanding how intertidal molluscs cope with short-term oxygen variations and gives useful biomarkers for environmental monitoring of hypoxic areas that are predicted to occur in the next future.

Diet and prey selectivity of the red mullet, Mullus barbatus (Pisces: Mullidae), from the southern Tyrrhenian Sea: the role of the surf zone as a feeding ground

Abstract The diet and prey selectivity of the red mullet, Mullus barbatus, on a sandy bottom of the Tyrrhenian Sea were examined, in respect of season, size and depth, focusing on the use of the surf zone as a feeding ground. Stomach contents of 99 specimens collected by beach seine (in the surf zone) and 568 specimens collected by trawl net (at 10, 20 and 30 m depths) were analysed. The overall diet of M. barbatus was dominated by crustaceans, mainly gammarids, while molluscs and polychaetes are secondary prey taxa. The diet composition of red mullet at each specific depth was related to the availability of prey in the benthic community: mysids were mostly preyed on in the surf zone, while gammarids, decapods and copepods were the principal crustacean prey organisms at other investigated depths. Red mullet in the surf zone mainly preyed on mysids and bivalves in summer and on cumaceans, gammarids and polychaetes in winter. At the other depths, seasonal variation in diet composition reflected the fluctuations of gammarid abundance in the environment; no differences in diet by size were detected. As a result, M. barbatus in the southern Tyrrhenian Sea can be considered as an opportunistic predator of benthic organisms, relying predominantly on crustaceans.