Marco Munari

First evidence of immunomodulation in bivalves under seawater acidification and increased temperature

Water acidification, temperature increases and changes in seawater salinity are predicted to occur in the near future. In such a global climate change (GCC) scenario, there is growing concern for the health status of both wild and farmed organisms. Bivalve molluscs, an important component of coastal marine ecosystems, are at risk. At the immunological level, the ability of an organism to maintain its immunosurveillance unaltered under adverse environmental conditions may enhance its survival capability. To our knowledge, only a few studies have investigated the effects of changing environmental parameters (as predicted in a GCC scenario) on the immune responses of bivalves. In the present study, the effects of both decreased pH values and increased temperature on the important immune parameters of two bivalve species were evaluated for the first time. The clam Chamelea gallina and the mussel Mytilus galloprovincialis, widespread along the coast of the Northwestern Adriatic Sea, were chosen as model organisms. Bivalves were exposed for 7 days to three pH values (8.1, 7.7 and 7.4) at two temperatures (22 and 28°C). Three independent experiments were carried out at salinities of 28, 34 and 40 PSU. The total haemocyte count, Neutral Red uptake, haemolymph lysozyme activity and total protein levels were measured. The results obtained demonstrated that tested experimental conditions affected significantly most of the immune parameters measured in bivalves, even if the variation pattern of haemocyte responses was not always linear. Between the two species, C. gallina appeared more vulnerable to changing pH and temperature than M. galloprovincialis. Overall, this study demonstrated that climate changes can strongly affect haemocyte functionality in bivalves. However, further studies are needed to clarify better the mechanisms of action of changing environmental parameters, both individually and in combination, on bivalve haemocytes.

Can the combination of decreased pH and increased temperature values induce oxidative stress in the clam Chamelea gallina and the mussel Mytilus galloprovincialis

The combined effects of decreased pH and increased temperature values on antioxidant enzyme activities and lipid peroxidation were evaluated for the first time in the clam Chamelea gallina and the mussel Mytilus galloprovincialis, two bivalve species that are widespread along the northwestern coast of the Adriatic Sea. For 7 days, bivalves were exposed to three pH values (8.1, 7.7 and 7.4) at two temperatures (22 and 28 °C). Three independent experiments were carried out at salinity values of 28, 34 and 40 psu. Superoxide dismutase, catalase and glutathione S-transferase activities as well as lipid peroxidation were measured in the gills and digestive gland of the bivalves. The results demonstrated that the experimental conditions significantly influenced the biochemical parameters of the bivalves, although the variation pattern varied depending on the species and tissues analysed.

Effects of the antidepressant fluoxetine on the immune parameters and acetylcholinesterase activity of the clam Venerupis philippinarum

Fluoxetine is an antidepressant used worldwide for the treatment of depression and other psychological disorders. The occurrence of fluoxetine in aquatic environments has been demonstrated. However, there is a lack of information about the effects of fluoxetine on non-target species, such as bivalve molluscs. In the present study, the effects of fluoxetine on the immune parameters of the clam Venerupis philippinarum were evaluated for the first time. Clams were exposed to various sublethal concentrations of fluoxetine (0, 1, 5, 25, 125, 625 μg l⁻¹) for 7 days, and the effects on the total haemocyte count (THC), the diameter and volume of haemocytes, haemocyte proliferation, Neutral Red uptake (NRU), and lysozyme activity in cell-free haemolymph (CFH) were evaluated. In addition, acetylcholinesterase (AChE) activity was measured in clam gills as a biomarker of neurotoxicity. A significant increase in THC values was observed in clams exposed to 25 μg l⁻¹ compared with controls, whereas no significant variations were recorded in either the diameter or the volume of haemocytes. Haemocyte proliferation increased significantly in animals exposed to 25, 125 and 625 μg l⁻¹ compared with controls. NRU decreased significantly in the haemocytes of clams exposed to 1 or 5 μg l⁻¹, whereas NRU returned to control values in clams exposed to the highest fluoxetine concentrations tested (25-625 μg l⁻¹). No significant alterations were observed in CFH lysozyme activity, whereas gill AChE activity decreased significantly in clams exposed to 1 or 5 μg l⁻¹. Overall, the obtained results demonstrated that fluoxetine markedly affected immune parameters and AChE activity in clams.

Combined effects of temperature and salinity on functional responses of haemocytes and survival in air of the clam Ruditapes philippinarum.

The combined effects of temperature and salinity on both immune responses and survival in air of the clam, Ruditapes philippinarum, were evaluated for the first time. The animals were kept for 7 days at three differing temperature (5 °C, 15 °C, 30 °C) and salinity values (18 psu, 28 psu, 38 psu), and effects of the resulting 9 experimental conditions on total haemocyte count (THC), Neutral Red uptake (NRU), haemolymph protein concentration, and lysozyme activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. The survival-in-air test was also performed. Two-way ANOVA analysis revealed that temperature influenced significantly THC and NRU, whereas salinity and temperature/salinity interaction affected NRU only. Temperature and salinity did not influence significantly HL and CFH lysozyme activity, as well as haemolymph total protein content. Survival-in-air test is widely used to evaluate general stress conditions in clams. In the present study, temperature and salinity were shown to influence the resistance to air exposure of R. philippinarum. The highest LT₅₀ (air exposure time resulting in 50% mortality) value was recorded in clams kept at 18 psu and 15 °C, whereas the lowest value was observed in clams kept at 28 psu and 30 °C. Overall, results obtained demonstrated that temperature and salinity can affect some functional responses of haemocytes from R. philippinarum, and suggested a better physiological condition for animals kept at 15 °C temperature and 18 psu salinity.

Does seawater acidification affect survival, growth and shell integrity in bivalve juveniles?

Anthropogenic emissions of carbon dioxide are leading to decreases in pH and changes in the carbonate chemistry of seawater. Ocean acidification may negatively affect the ability of marine organisms to produce calcareous structures while also influencing their physiological responses and growth. The aim of this study was to evaluate the effects of reduced pH on the survival, growth and shell integrity of juveniles of two marine bivalves from the Northern Adriatic sea: the Mediterranean mussel Mytilus galloprovincialis and the striped venus clam Chamelea gallina. An outdoor flow-through plant was set up and two pH levels (natural seawater pH as a control, pH 7.4 as the treatment) were tested in long-term experiments. Mortality was low throughout the first experiment for both mussels and clams, but a significant increase, which was sensibly higher in clams, was observed at the end of the experiment (6 months). Significant decreases in the live weight (-26%) and, surprisingly, in the shell length (-5%) were observed in treated clams, but not in mussels. In the controls of both species, no shell damage was ever recorded; in the treated mussels and clams, damage proceeded via different modes and to different extents. The severity of shell injuries was maximal in the mussels after just 3 months of exposure to a reduced pH, whereas it progressively increased in clams until the end of the experiment. In shells of both species, the damaged area increased throughout the experiment, peaking at 35% in mussels and 11% in clams. The shell thickness of the treated and control animals significantly decreased after 3 months in clams and after 6 months in mussels. In the second experiment (3 months), only juvenile mussels were exposed to a reduced pH. After 3 months, the mussels at a natural pH level or pH 7.4 did not differ in their survival, shell length or live weight. Conversely, shell damage was clearly visible in the treated mussels from the 1st month onward. Monitoring the chemistry of seawater carbonates always showed aragonite undersaturation at 7.4 pH, whereas calcite undersaturation occurred in only 37% of the measurements. The present study highlighted the contrasting effects of acidification in two bivalve species living in the same region, although not exactly in the same habitat.

Genotoxic effects of CdS quantum dots and Ag2S nanoparticles in fish cell lines (RTG-2)

The increasing use of nanotechnologies will lead to significant releases of engineered nanoparticles into the aquatic environment, where their impact is still poorly characterized. In the present paper, the genotoxic and cytotoxic properties of CdS quantum dots (QDs) and silver sulphide (Ag2S) coated with methyl polyethylene glycol (M-PEG) were investigated in a rainbow trout cell line (RTG-2). The results showed that CdS QDs were highly cytotoxic at high concentrations (10 and 50μg/ml), and exhibited a concentration-dependent genotoxicity in the sub-toxic range (0.01-1μg/ml) after 24h exposure. Ag2S showed neither genotoxic nor cytotoxic effects.

Genotoxic effects of Ag2S and CdS nanoparticles in blue mussel (Mytilus edulis) haemocytes

The use of functionalised metal sulphide nanoparticles (NPs) for nanoremediation and biomedical application is rapidly increasing, which could lead to significant inputs into the marine environment. The potential impact of some NPs on marine organisms is still poorly understood. In the present paper the genotoxic potential of Ag2S and CdS NPs on Mytilus edulis haemocytes was assessed. MPEG-SH (thiol-terminated methyl polyethylene glycol), was used as capping agent to avoid NPs agglomeration. TEM analysis showed that the Ag2S NPs size was 13±7 nm, whereas CdS quantum dots had an average diameter of 4±1 nm. DNA integrity was evaluated by Comet assay following exposure to increasing concentration series (0.01–10 mg/L). Both silver and cadmium NPs showed genotoxic effects at the highest dose. MPEG-SH was also found to exert a weak genotoxic activity, suggesting that at least part of the genotoxic potential of functionalised NPs on mussel cells might be attributable to the capping agent. These results confirm the genotoxic potential of Ag2S NPs for mussel cells and demonstrated, for the first time, that CdS NPs is genotoxic in a marine organism.

L'Aquila 6th April 2009 Earthquake: Emergency and Post-emergency Activities on Cultural Heritage Buildings

The earthquake that struck the Abruzzo region on 6th April 2009 at 3:32 a.m., had its epicentre in the capital of the region, L’Aquila, and seriously affected a wide area around the city, where many historic towns and villages are found. Due to the strategic importance of L’Aquila, a strong and organized civil protection action was necessary to face the emergency. In addition, the structural damage on historic buildings and centres was enormous. Therefore, it was necessary to carry out specific actions aimed at the safeguard of this heritage. These can be shortly listed as: set up of the organizational and decisional structure, damage surveys, temporary interventions to provide the minimum safety conditions, set up of a monitoring plan for some important monuments, set up of a methodology to intervene on complex and connected buildings in the historic centres, definition of adequate materials and techniques to intervene on the damaged buildings.

Cultural Heritage Buildings and the Abruzzo Earthquake: Performance and Post-Earthquake Actions

The architectural heritage was seriously hit by the earthquake that occurred on April 6th 2009 in the Abruzzo region, especially considering the effects on a city with the size and with historical and strategic importance as a capital of a region, L’Aquila. The activities to protect that heritage have been centralized in the structure “Protection of Cultural Heritage” at Di.Coma.C. (Command and Control Quarter), managed by the Civil Protection Department. This allowed the cooperation among different involved subjects (Ministry of Cultural Heritage officers, experts on structural engineering from Universities and Fire Brigade teams), with their own specific knowledge. Keystone of the operating process was the standardization of the damage survey and of its immediate and correct interpretation, through dedicated survey forms for churches and palaces. The experience in the field of temporary safety measures was extremely interesting: ideas for engineering the process were developed, in cooperation with the work of the fire brigade men, that are highly experienced technicians in the “emergency” field. Finally, monitoring plans for some important monuments have been set up for the control of the damage progression and the analysis of the structural behavior of buildings after the earthquake and the execution of temporary interventions: two cases (St. Mark Church and the Spanish Fortress) are discussed.

Strengthening of Stone and Brick Masonry Buildings

Today, the scientific community has recognised that the structural safety aspects of existing masonry buildings cannot be treated according to standard procedures that are fit for new constructions. Hence, new approaches for assessing the actual structural performance of existing masonry buildings and developing more appropriate methods and criteria for their repair and strengthening are in progress. The basic idea is that the usual design approaches naturally imply a certain level of “over-design”, and this can lead to unacceptable solutions, under the point of view of costs and conservation, when dealing with existing structures. For these reasons, attention must be paid to the appropriate selection and design of materials and technologies for intervention, taking into account the possibilities offered by traditional solutions and their possible combinations with innovative ones. As existing buildings are usually designed for vertical actions, the “seismic conditions” have the most awkward implications. Indeed, the earthquake actions refer to the very extreme structural resources, i.e. those connected with resistant mechanisms that are normally neglected, and are very difficult to be implemented into structural models. In this chapter, after a general introduction on the characteristics and peculiarities of existing buildings made of stone and clay brick masonry, where we will take into account mainly ordinary buildings, and after some general considerations on the more suitable approaches and criteria for the design of interventions, the most relevant techniques used to strengthen this kind of buildings will be presented.