Epidemic Mortality of the Sponge Ircinia variabilis (Schmidt, 1862) Associated to Proliferation of a Vibrio Bacterium

In recent years, several episodes of mass mortality of sessile epibenthic invertebrates, including sponges, have been recorded worldwide. In the present study, we report a disease event on Ircinia variabilis recorded in September 2009 along the southern Adriatic and Ionian seas (Apulian coast), with the aim to quantify the mortality incidence on the sponge population, to investigate the effect of the disease on the sponge tissues and to assess whether the disease is associated with vibrios proliferation. The injured sponges showed wide necrotic areas on the surface or disruption of the body in several portions. Necrotic areas were whitish and often were covered with a thin mucous coat formed by bacteria. In the most affected specimens, sponge organisation resulted partial or complete loss, with the final exposure of the dense skeletal network of spongine fibres to the environment. The results of microbiological cultural analysis using in parallel Marine Agar 2216 and thiosulphate/citrate/bile salts/sucrose agar demonstrated that, in affected specimens, vibrios represented 15.8 % of the total I. variabilis surface culturable bacteria. Moreover, all the isolated vibrios, grown from the wide whitish areas that characterize the surface of the diseased sponges, were identified, and their assignment to the Vibrio rotiferianus was consistent with phylogenetic analysis and data of morphological, cultural and biochemical tests. Studies on V. rotiferianus have shown that its pathogenicity, with respect to various aquatic organisms, is higher than that of Vibrio harveyi. The factors triggering the disease outbreak in Ircinia variabilis populations remain unclear. At present, we can hypothesize the involvement in the disease of a synergetic mechanism that, under stressful physiological conditions (high temperature, elevated nutrients and reduced water flow), induces sponge pathogens, in our case V. rotiferanius, to become virulent, making sponges unable to control their proliferation. Additional studies are needed to understand the etiological processes as well as the factors involved in sponges recovering from this epidemic event allowing them to face mass mortality. A drastic reduction of sponge-specific representatives could have marked a negative impact on the environmental health on account of their role in the sea remediation processes as filter-feeding organisms.     

The spermatodesm of Cloeon dipterum (L.): fine structure and sperm movement

Sperm bundles of the ephemeropteran Cloeon dipterum (L.) were found in the seminal vesicle. They are apically protected by cyst cells and sperm anterior regions are embedded in a secretion produced by these cells. Cyst cells further degenerate making sperm cells free. The sperm bundles are able to perform a forward progression only when sperm are stick together with their tail posterior tips. On the contrary, when sperm have their tail ends free, they beat but are unable to progress. The functional significance of the sperm bundles is discussed.     

Electrophysiological identification of thermo- and hygro-sensitive receptor neurons on the antennae of the dragonfly Libellula depressa

Recent ultrastructural investigations on Odonata antennal flagellum describe two types of sensilla styloconica, T1 and T2. The styloconic sensilla are located in pits, at the bottom of deep cavities, and share common features typical of thermo-hygroreceptors. In order to ascertain if the Odonata antennae are involved in hygroreception and thermoreception, we carried out electrophysiological recordings (single cell recordings, SCR) from adult males and females of Libellula depressa L., 1758. After contact was established, the antenna was stimulated by rapid changes in temperature and humidity. The present research shows the occurrence of a dry (DC), a moist (MC) and a cold (CC) receptor neurons on the antennal flagellum of L. depressa. These data demonstrate for the first time the presence of functional thermo-hygroreceptors on the antennal flagellum of dragonflies. The present results extend our knowledge of the not visual sensory modalities of Odonata, a field of research unexplored so far.     

The gustatory sensilla on the endophytic ovipositor of Odonata

The present paper aims at describing the fine structure of coeloconic sensilla located on the cutting valves of the endophytic ovipositor of two Odonata species, the anisopteran Aeshna cyanea (Aeshnidae) and the zygopteran Ischnura elegans (Coenagrionidae), by carrying out parallel investigations under SEM and TEM. In both species these coeloconic sensilla are innervated by four unbranched neurons forming four outer dendritic segments enveloped by the dendrite sheath. One dendrite terminates at the base of the peg forming a well developed tubular body, while the other three enter the peg after interruption of the dendrite sheath. The cuticle of the peg shows an apical pore and a joint membrane. This last feature, together with the tubular body and the suspension fibers, represent the mechanosensory components of the sensillum while the pore and the dendrites entering the peg allow chemoreception. The ultrastructural organization of these coeloconic sensilla is in agreement with the one reported for insect gustatory sensilla. Our investigation describes for the first time typical insect gustatory sensilla in Odonata. Electrophysiological and behavioral studies are needed to verify the role that these structures can perform in sensing the egg-laying substrata.     

Identification of Relevant Conformational Epitopes on the HER2 Oncoprotein by Using Large Fragment Phage Display (LFPD)

We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines.     

Snake venomics of Crotalus tigris: the minimalist toxin arsenal of the deadliest Neartic rattlesnake venom. Evolutionary Clues for generating a pan-specific antivenom against crotalid type II venoms

We report the proteomic and antivenomic characterization of Crotalus tigris venom. This venom exhibits the highest lethality for mice among rattlesnakes and the simplest toxin proteome reported to date. The venom proteome of C. tigris comprises 7-8 gene products from 6 toxin families; the presynaptic β-neurotoxic heterodimeric PLA(2), Mojave toxin, and two serine proteinases comprise, respectively, 66 and 27% of the C. tigris toxin arsenal, whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1-2 PIII-SVMPs each represent 0.1-5% of the total venom proteome. This toxin profile really explains the systemic neuro- and myotoxic effects observed in envenomated animals. In addition, we found that venom lethality of C. tigris and other North American rattlesnake type II venoms correlates with the concentration of Mojave toxin A-subunit, supporting the view that the neurotoxic venom phenotype of crotalid type II venoms may be described as a single-allele adaptation. Our data suggest that the evolutionary trend toward neurotoxicity, which has been also reported for the South American rattlesnakes, may have resulted by pedomorphism. The ability of an experimental antivenom to effectively immunodeplete proteins from the type II venoms of C. tigris, Crotalus horridus , Crotalus oreganus helleri, Crotalus scutulatus scutulatus, and Sistrurus catenatus catenatus indicated the feasibility of generating a pan-American anti-Crotalus type II antivenom, suggested by the identification of shared evolutionary trends among South and North American Crotalus species.     

Isolation of oryzanol from crude rice bran oil

The isolation of oryzanol from crude rice bran oil (RBO) was achieved by a two-step crystallization process. In the first crystallization, oryzanol was concentrated in the liquid phase along with free fatty acid (FFA), monoacylglycerol (MG), squalene, tocols, and phytosterols, whereas the solid phase contained mainly triacylglycerol (TG) and steryl esters. Oryzanol-rich product obtained from the first crystallization was subjected to the second crystallization where the oryzanol-rich product was kept at room temperature (20.5+/-1.5 degrees C) for 24h. Hexane was added as anti-solvent to the oryzanol-rich product and kept at 5+/-1 degrees C for another 48h. Parameters that affect the isolation of oryzanol from crude RBO were systematically investigated. Under optimal operation conditions, oryzanol with purity and recovery of 93-95% and 59%, respectively, was obtained from RBO with an initial FFA content of ca. 5%.     

A phase II trial of vaccination with autologous, tumor-derived heat-shock protein peptide complexes Gp96, in combination with GM-CSF and interferon-α in metastatic melanoma patients

The aim of this study was to determine the immunogenicity and antitumor activity of autologous, tumor-derived heat shock protein gp96-peptide complex vaccine (HSPPC-96; Oncophage®) given with GM-CSF and IFN-α in pre-treated metastatic (AJCC stage IV) melanoma patients. Patients underwent surgical resection of metastatic lesions for HSPPC-96 production. HSPPC-96 was administered subcutaneously (s.c.) in four weekly intervals (first cycle). Patients with more available vaccine and absence of progressive disease received four additional injections in 2-week intervals (second cycle) or more. GM-CSF was given s.c. at the same site at days –1, 0 and +1, while IFN-α (3 MU) was administered s.c. at a different site at days +4 and +6. Antigen-specific anti-melanoma T and NK lymphocyte response was assessed by enzyme-linked immunospot assay on peripheral blood mononuclear cells obtained before and after vaccination. Thirty-eight patients were enrolled, 20 received at least four injections (one cycle) of HSPPC-96 and were considered assessable. Toxicity was mild and most treatment-related adverse events were local erythema and induration at the injection site. Patients receiving at least four injections of HSPPC-96 were considered evaluable for clinical response: of the 18 patients with measurable disease post surgery, 11 showed stable disease (SD). The ELISPOT assay revealed an increased class I HLA-restricted T and NK cell-mediated post-vaccination response in 5 out of 17 and 12 out of the 18 patients tested, respectively. Four of the five class I HLA-restricted T cell responses fall in the group of SD patients. Vaccination with autologous HSPPC-96 together with GM-CSF and IFN-α is feasible and accompanied by mild local and systemic toxicity. Both tumor-specific T cell-mediated and NK cell responses were generated in a proportion of patients. Clinical activity was limited to SD. However, both immunological and clinical responses were not improved as compared with those recorded in a previous study investigating HSPPC-96 monotherapy.L.P. and R.P. have equally contributed to the work.