Maresca, Francesca (2026) Investigating Mass Mortality Events in Mediterranean benthic species: from antibody validation to case studies. [Tesi di dottorato]

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Abstract

Mass mortality events (MMEs) have increasingly affected marine organisms worldwide, with severe consequences for benthic species in the Mediterranean Sea. These events are the result of complex and multifactorial processes rather than caused by a single causative agent, in which environmental stressors, host physiological condition and biological factors interact synergistically, bringing to a disease condition. However, despite their growing ecological relevance, the investigation of these phenomena is often hampered by limitations in non-model marine species. This work starts aiming to improve the characterization of the aetio-pathogenetic component associated with MMEs in some selected Mediterranean marine benthic species, including both vertebrates (Mullus barbatus) and invertebrates (Paracentrotus lividus, Mytilus galloprovincialis and Pinna nobilis), through the research, validation and application of tissue markers useful for a more comprehensive understanding of the whole pathogenic process. With this purpose, antibody validation of targeting proteins involved in cellular stress responses (Hsp70, Hsp90), apoptosis (pro-Caspase 3 and active Caspase 3), genotoxic stress (p53) and detoxification and contaminant exposure (metallothioneins and Cyp1α) was performed by Western Blot using commercially available mammalian antibodies. This approach was needed to assess antibody cross-reactivity and specificity in phylogenetically distant taxa, such as fish, echinoderms and bivalves, prior to their application in immunohistochemistry. Following this, the validated markers were subsequently applied in monitoring approach and disease studies to highlight their potential use in describing the host responses during a morbid condition. A comparison of the antibody used in BLASTp database indicated differences and similarities for molecular chaperones and apoptotic proteases, moderate/scarce evolutionary conservation for the transcription factor such as p53 and the cytochrome Cyp1α, and high diversification in invertebrates for metallothioneins (MT), that are an extremely heterogeneous superfamily evolved through a web of duplication, functional differentiation and convergence events. In collaboration with ISPRA institute (Istituto Superiore per la Protezione e la Ricerca Ambientale) in the framework of the Marine Strategy Framework Directive, the tissue markers Cyp1α and MT were integrated with biochemical and molecular biomarkers and animal histopathology to improve the interpretation and understanding of the biological individual effects related to contaminant exposure in the sentinel species Mullus barbatus. The study highlighted the triggering of the biotransformation pathway along with the presence of histopathological lesions and parasitic infections helping in biomarkers interpretation. The chaperones markers related to cellular stress (Hsp70 and Hsp90) were used to investigate the host response associated with the Bald Sea Urchin Disease in a natural population from the Gulf of Naples of the Sea Urchin Paracentrotus lividus. The study showed the immune-signalling cascades that are linked to cellular stress responses, with the local accumulation of Hsp70 in cytoplasm of the amoebocytes, likely stimulated during the BSUD by an accumulation of unfolded proteins. This mechanism might serve as a signal amplification cascade favouring cytokine production. The Hsp70 used, the isoform HspA1A, was also expressed in the basal membrane of digestive epithelia and epidermis, spines, pedicellaria and tube feet of cytoskeleton in both symptomatic and asymptomatic individuals, underlying its inducible but also constitutive basal activity. Moreover, we showed that BSUD has a complex multifactorial aetiology, driven by environmental stress and microbial components that involved Vibrio species. Intestinal parasite of marine bivalves can cause direct mortalities and morbidity, with consequent poor growth, higher susceptibility to opportunistic pathogens and lower resistance to stress. The pathogens that invade the gastrointestinal tract can elicit a strong inflammatory reaction (concerted by cytokines) that can have fatal pathophysiological consequence. In shellfish belonging to different group some of these pathogens are part of the WHOA listed diseases, common in farmed species, like Marteliosis and Perkinsosis, or present in wild populations and be related to MMEs, like the Haplosporidiosis. In this study, the multiple application of Hsp70 and active Caspase-3, coupled with histopathology, allowed a better characterization of host stress response and activation of apoptosis at tissue level. Active caspase-3 was detected in the epithelial cells of the digestive epithelia for two of the studied pathogens. The labelling appeared to be present at the level of the lysosomes in the A cells of digestive gland where caspase-3 activation is closely connected, likely causing the leakage of cathepsins and other proteases that can directly activate pro-caspase-3 in the cytosol, or autolysosomes. The process involves lysosomal dysfunction, leading to enzyme release, which triggers the caspase cascade and final cell death. At the same time, this mechanism, along with the activation of Hsp70 in the first part of the digestive tract, enables tissue protection with the activation of cellular stress, during the infection. Overall, the results of this study demonstrate that validated tissue markers, coupled with other diagnostic approaches (i.e. histopathology, scanning electron microscopy, molecular biology and biomarker analyses) represent reliable tools for investigating morbid state in marine organisms, providing a more robust interpretation of the pathogenic processes across different taxa and a more comprehensive approach useful for environmental monitoring and conservation strategies in the Mediterranean Sea.

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