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The increasing use of pharmaceuticals in human and veterinary medicine, along with their poor removal rates at wastewater treatment facilities is resulting in the chronic release of pharmaceutically-active compounds (PhACs) into the marine environment, where they pose a threat to non-target organisms. A useful approach, as applied in the current study for assessing the effects of PhACs on non-target organisms, is the proteomic approach: the large-scale study of an organism's proteins. Using ‘shotgun’ proteomics, we identified differentially-expressed proteins based on peptide fragments in the solitary ascidian, Herdmania momus, following a 14-day laboratory experimental exposure to the PhAC carbamazepine (CBZ), an anticonvulsant and antidepressant medication, frequently detected in the aquatic environment. Individuals were exposed to environmentally relevant concentrations: 5 or 10 µg/L of CBZ, in addition to a control treatment. Out of 199 identified proteins, 24 were differentially expressed (12%) between the treatment groups, and thus can potentially be developed as biomarkers for CBZ contamination. Ascidians’ phylogenetic position within the closest sister group to vertebrates presents an advantage in examining the pathological effects of PhACs on vertebrate-related organs and systems. Together with the world-wide distribution of some model ascidian species, and their ability to flourish in pristine and polluted sites, they provide a promising tool through which to study the extent and effects of PhAC contamination on marine organisms.

Ascidians (Chordata, Ascidiacea) are considered to be prominent marine invaders, able to tolerate highly polluted environments and fluctuations in salinity and temperature. Here, we examined the seasonal and spatial dynamics of the microbial communities in the inner-tunic of two invasive ascidians, Styela plicata (Lesueur 1823) and Herdmania momus (Savigny 1816), in order to investigate the changes that occur in the microbiome of non-indigenous ascidians in different environments. Microbial communities were characterized using next-generation sequencing of partial (V4) 16S rRNA gene sequences. A clear differentiation between the ascidian-associated microbiome and bacterioplankton was observed, and two distinct sets of operational taxonomic units (OTUs), one core and the other dynamic, were recovered from both species. The relative abundance of the dynamic OTUs in H. momus was higher than in S. plicata, for which core OTU structure was maintained independently of location. Ten and seventeen core OTUs were identified in S. plicata and H. momus, respectively, including taxa with reported capabilities of carbon fixing, ammonia oxidization, denitrification, and heavy-metal processing. The ascidian-sourced dynamic OTUs clustered in response to site and season but significantly differed from the bacterioplankton community structure. These findings suggest that the associations between invasive ascidians and their symbionts may enhance host functionality while maintaining host adaptability to changing environmental conditions.

Peptide epitopes presented through class I major histocompatability complex (MHC class I) on the cell surface, are generated by proteolytic processing of protein-antigens in the cytoplasm. The length and amino acid sequence determine whether a given peptide can fit into the peptide binding groove of class I heavy chain molecules and subsequently be presented to the immune system. The mode of action of the processing pathway is therefore of great interest. To study the processing mechanism of MHC class I-restricted intracellular antigens, we reconstituted the proteolytic processing of a model antigen in a cell-free system. Incubation of oxidized and urea-treated OVA in lymphocyte lysate resulted in partial degradation of the antigen. Degradation of the antigen depended on the presence of ATP. Addition of methylated ubiquitin abolished the reaction which was then restored by addition of an excess of native ubiquitin, indicating that the breakdown of the antigen in lymphocyte lysate is mediated by the ubiquitin proteolytic system. Upon incubation of modified OVA in lymphocyte lysate, a specific antigenic peptide was generated. The peptide was recognized by cytotoxic T lymphocytes directed against OVA-derived, H-2Kb-restricted peptide (SIINFEKL), and by a monoclonal antibody that recognizes cell-bound Kb-SIINFEKL complexes. Formation of the peptide epitope depended on the presence of ATP and ubiquitin. These results indicate that proteolytic processing of modified OVA is carried out by the ubiquitin-mediated degradation system. The experimental system described provides a tool to analyze the molecular mechanisms underlying the generation of specific, MHC class I-restricted peptide epitopes.