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This study examined the effects of boat noise on the behavioural and biochemical parameters of the Mediterranean spiny lobster (Palinurus elephas). The experiment was conducted in a tank equipped with a video and audio recording system. 18 experimental trials, assigned to boat noise and control conditions, were performed using lobsters in single and group of 4 specimens. After a 1h habituation period, we audio- and video-recorded the lobsters for 1h. During the experimental phase, the animals assigned to the boat groups were exposed to boat noise pollution (a random sequence of boat noises). Exposure to the noise produced significant variations in locomotor behaviours and haemolymphatic parameters. Our results indicate that the lobsters exposed to boat noises increased significantly their locomotor activities and haemolymphatic bioindicator of stressful conditions such as glucose, total proteins, Hsp70 expression and THC when tested both singly and in groups.

Recently described biochemical and structural aspects of fucose-binding lectins from the European eel (Anguilla anguilla) and striped bass (Morone saxatilis) led to the identification of a novel lectin family ("F-type" lectins) characterized by a unique sequence motif and a characteristic structural fold. The F-type fold is shared not only with other members of this lectin family, but also with apparently unrelated proteins ranging from prokaryotes to vertebrates. Here we describe the purification, biochemical and molecular properties, and the opsonic activity of an F-type lectin (DlFBL) isolated from sea bass (Dicentrarchus labrax) serum. DlFBL exhibits two tandemly arranged carbohydrate-recognition domains that display the F-type sequence motif. In situ hybridization and immunohistochemical analysis revealed that DlFBL is specifically expressed and localized in hepatocytes and intestinal cells. Exposure of formalin-killed Escherichia coli to DlFBL enhanced their phagocytosis by D. labrax peritoneal macrophages relative to the unexposed controls, suggesting that DlFBL may function as an opsonin in plasma and intestinal mucus.

The present study aims to investigate coelomocytes, immune mediators cells in the echinoderm Holothuria tubulosa, as an unusual source of antimicrobial and antibiofilm agents. The activity of the 5kDa peptide fraction of the cytosol from H. tubulosa coelomocytes (5-HCC) was tested against a reference group of Gram-negative and Gram-positive human pathogens. Minimal inhibitory concentrations (MICs) ranging from 125 to 500 mg/ml were determined against tested strains. The observed biological activity of 5-HCC could be due to two novel peptides, identified by capillary RP-HPLC/nESI-MS/MS, which present the common chemical-physical characteristics of antimicrobial peptides. Such peptides were chemically synthesized and their antimicrobial activity was tested. The synthetic peptides showed broad-spectrum activity at 12.5 mg/ml against the majority of the tested Gram-positive and Gram-negative strains, and they were also able to inhibit biofilm formation in a significant percentage at a concentration of 3.1 mg/ml against staphylococcal and Pseudomonas aeruginosa strains.The immune mediators in H. tubulosa are a source of novel antimicrobial peptides for the development of new agents against biofilm bacterial communities that are often intrinsically resistant to conventional antibiotics.

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SUMMARYThis study examined the effects of an acoustic stimulus on the haemolymph and agonistic behaviour of the red swamp crayfish, Procambarus clarkii. The experiment was conducted in a tank equipped with a video recording system using six groups (three control and three test groups) of five adult crayfish (30 specimens in total). After 1h of habituation, the behaviour of the crayfish was monitored for 2h. During the second hour, the animals in the test groups were exposed to a linear sweep (frequency range 0.1-25kHz; peak amplitude 148dB rms re.1μPa at 12kHz) acoustic stimulus for 30min. Exposure to the noise produced significant variations in haemato-immunological parameters as well as a reduction in agonistic behaviour.

A tumor necrosis factor-alpha (TNFα)-like gene from Ciona intestinalis (CiTNFα-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation. An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFα-like cytokine was upregulated by in vivo LPS challenge supporting its proinflammatory role. In the pharynx, increased expression levels were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human TNFα epitopes in a 15-kDa protein component of the hemolymph serum and in a 43-kDa protein contained in the hemocyte lysate supernatant prepared in the presence of detergents. Both soluble and hemocyte-bound CiTNFα-like protein therefore appeared to be modulated by the LPS challenge.

Studies on inducible ascidian lectins may shed light on the evolutionary emergence of cytokine functions. Here, we show that the levels of opsonins, with IL1alpha-epitopes, increase in Ciona intestinalis hemolymph as a response to an inflammatory stimulus and, in particular, to intratunic injection of lipopolysaccharide (LPS). The inflammatory agent promptly (within 4 h) enhances Ca(2+)-independent serum hemagglutinating and opsonizing activities, which are both inhibited by D-galactose and D-galactosides (alpha-lactose, N-acetyl-D-lactosamine, thio-digalactoside), suggesting that anti-rabbit erythrocyte lectins with galectin properties are involved as opsonins. Inducible galectin molecules contain interleukin-1alpha (IL1alpha) epitopes, and their activities are specifically inhibited by anti-human recombinant IL1alpha antibody. Analysis by SDS-polyacrylamide gel electrophoresis has revealed that the density of the bands of several serum proteins increases within 4 h after LPS injection, correlated with the enhanced serum activity. Moreover, Western blot patterns demonstrate that several serum proteins (59, 37, 30, 23, 15 kDa) cross-react with the antibody as early as 4 h post-injection. Although we have not been able to establish whether, in adition to galectins, various types of D-galactose-specific lectins are contained in the serum, we show, for the first time in invertebrates, that galectin molecules with opsonic properties can be enhanced in response to a non-specific inflammatory stimulus, and that their release can be further stimulated by LPS. Finally, we reveal that multiple galectins share human IL1alpha epitopes, probably because of steric configuration and the oligomerization process.