Maria Rosa Trapani

Evaluation of waterborne exposure to heavy metals in innate immune defences present on skin mucus of gilthead seabream (Sparus aurata).

Aquatic animals are continuously exposed to chemical pollutants but the effects evoked in skin surfaces, which receive the most direct contact with them, are poorly investigated. Terminal carbohydrate composition and immunological components present in skin mucus of gilthead seabream (Sparus aurata L.) specimens exposed to waterborne sublethal dosages of heavy metals [arsenic (As2O3), cadmium (CdCl2) and mercury (CH3HgCl) at 5, 5 and 0.04 μM, respectively for 2, 10 and 30 days were analysed. Moreover, the presence of a fucose binding lectin (FBL) was evaluated by western blot and the protein profiles were by SDS-PAGE and HPLC. Results showed little effects of heavy metals in the presence of several terminal carbohydrates with few increments or decrements. Most of the enzyme activities related to immune responses were increased upon heavy metal exposure in the skin mucus including bactericidal activity. Methylmercury produced the most dramatic changes increasing all the activities. Moreover, the FBL was undetected in any of the control fish skin mucus but was evident in all the heavy metal exposed fish. In addition, As and Cd produced a clear change in the protein profile as evidenced by the lack of a protein band of around 12 kDa which is absent. These protein changes were more evident with the HPLC study showing the presence of different peaks and differences in intensity. The present results could be useful for better understanding the role and their behaviour of the mucosal immunity in skin as a key component of the innate immune system against pollutants.

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.

Upregulated transcription of phenoloxidase genes in the pharynx and endostyle of Ciona intestinalis in response to LPS.

We investigated the role of phenoloxidases (POs) in ascidians inflammatory reaction, a components of a copper-containing protein family involved in invertebrate immune system. In Ciona intestinalis two phenoloxidases (CinPO-1, CinPO-2) have been sequenced. In the present study, real time PCR analysis showed that both CinPO-1 and CinPO-2 genes were modulated by LPS inoculation suggesting that they are inducible and highly expressed in the inflamed pharynx. In situ hybridization disclosed CinPO-1 and CinPO-2 transcripts in pharynx hemocytes (granulocytes) and, mainly, in unilocular refractile granulocytes (URG) which mainly populated the inflamed tunic matrix. Interestingly, the genes are also upregulated by LPS in the endostyle (zones 7, 8 and 9) that is considered homolog to the vertebrate thyroid.

Marine organisms as source of bioactive molecules applied in restoration projects

In recent decades research in the conservation and restoration field has provided sustainable alternatives to traditional procedures for cleaning or controlling the microbial colonization of works of art. In the present study, for the first time novel bioactive molecules extracted from marine invertebrate organisms (Anthozoa) were tested instead of chemical compounds for removing protein layers or as a biocide for controlling fungal or bacterial colonization. In particular, Bioactive Molecules with Protease activity (BMP), acting in a temperature range of 4- 30°C, were tested for the hydrolysis of protein layers on laboratory specimens. The cleaning protocol provides a selective procedure to avoid damage to the original materials constituting the heritage object.Concurrently, enzymatic cleaning was also performed using commercial Protease from Aspergillus sojae (Type XIX), in order to compare their hydrolytic activities. Bioactive Molecules with Antimicrobial activity (BMA1, BMA2) were tested to control bacterial (Bacillus, Micrococcus) or fungal (Aspergillus, Penicillium) growth, previously isolated from colonized canvas samples and characterized by an integrated approach based on in vitro culture, microscopy and molecular investigations. These molecules were tested to define the Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal/ Fungicidal Concentration (MBC/MFC). Specifically, BMAs were used to control fungal growth during the relining of the painting (laboratory specimens), carried out using a canvas support, and glue paste as binder.In our hypothesis, these molecules provide an important contribution to the development of innovative protocols for biocleaning or microbial growth control, based on fast and easy application, operator friendly and environmentally sustainable molecules.

Innovative and Integrated Strategies: Case Studies

In this chapter, case studies related to biodeterioration, bioaerosol, biocide and biocleaning are reported. The aim is highlighting the role of biology and biotechnology tools for the preventive conservation of organic and inorganic artifacts, understanding how traditional as well as innovative methods can help the conservationists to develop integrated strategies considering works of art/environment/humans as a dynamic system. Particularly, based on the experience acquired during the researches of Laboratory of Biology and Biotechnology for Cultural Heritage (LaBBCH), the authors suggest several approaches to reveal and identify biological systems able to induce biodeterioration of cultural assets, also focusing on bioaerosols in indoor environment to assess the risk for historical-artistic collections. Finally, novel bioactive molecules have been applied to perform biocleaning protocols or to control of microbial colonisation, in accordance to conservative restoration procedures and safety for both the environment and operators.