Health status of aquacultured bivalves and fish: immune responses of Mytilus galloprovincialis to bacterial isolates and monitoring growth of Sparus aurata and Dicentrarchus labrax

This PhD project was carried out within the framework of the Italian National Biodiversity Future Center (NBFC), National Recovery and Resilience Plan (NRRP). The NRRP is the national program through which Italy used the European NextGenerationEU funds to promote economic recovery, innovation, sustainability, and social resilience after the COVID-19 crisis. The plan was organized into six missions, including digitalization, ecological transition, infrastructure, education and research, inclusion, and health. This project was enrolled within Mission 4 – Education and Research. This mission gave birth also to the National Biodiversity Future Center (NBFC), a national research centre funded to study, monitor, restore, and valorize biodiversity in Italy and the Mediterranean area. It involved dozens of universities, research centers, and private partners working together to develop scientific knowledge and technological solutions to address biodiversity loss and promote sustainable development. NBFC research topics were divided into 8 different spokes regarding: Sea (Spoke 1-2), Land (Spoke 3-4), Urban (Spoke 5-6) and Impact (Spoke 7-8). Within Spoke 2 (Marine biodiversity and sustainable management of marine resources), Activity 3 included research tasks aimed at improving sustainability, resilience, and animal welfare in aquaculture systems. This PhD thesis was enrolled in two relevant tasks belonging to Activity 3, Task 3.3 and Task 3.4, which focused on fish welfare and disease resilience respectively. Tasks 3.3 and 3.4 addressed sustainable aquaculture from complementary points of view: together they contribute to healthier aquaculture systems with lower disease incidence and reduced dependence on antimicrobials, supporting long-term sustainability. - Task 3.4 - Selective breeding for disease resistance and resilience to environmental challenges for sustainable fish and shellfish aquaculture: Responses of aquacultured bivalves to environmental stressors and potential pathogens - Task 3.3 - Better health and welfare of fish without antimicrobials: Assessment of fish welfare in different farming systems Therefore, within the planned activities, the first part of the research (Section I) is related to Task 3.4, and it is focused on the marine mussel Mytilus galloprovincialis, and on the role of immune system in the response to challenge with bacterial isolates from bivalve mortality events (Chapters 1-4). Chapter 1, 2 and 3 report data on a new potential bivalve pathogen isolated from a mortality event affecting adult M. galloprovincialis in 2022 in the Ebro Delta (Spain). Chapter 1: this bacterium was firstly identified as Vibrio ostreicida and a PCR protocol was developed for its detection in mussel tissues and water samples. These data represented the first evidence for the association of V. ostreicida with a mortality episode in mussels. Chapter 2: the immune interactions between a strain of V. ostreicida and bivalve hosts, the mussel M. galloprovincialis and the oyster Magallana gigas were evaluated. The results revealed distinct immune profiles, indicating species-specific differences in susceptibility to V. ostreicida. Chapter 3: the infection dynamics and immune responses of adult M. galloprovincialis challenged with V. ostreicida were investigated using different in vivo experimental approaches: injection/cohabitation experiments and bath infections. The results showed that M. galloprovincialis is able to cope with challenge with V. ostreicida, indicating that this Vibrio species is moderately pathogenic to mussels under the established experimental conditions. Chapter 4 reports data on the immune responses of M. galloprovincialis to polymicrobial infection with two bacteria previously isolated from an oyster mortality event occurred in the Ebro Delta: Vibrio aestuarianus strain 106 and Malaciobacter marinus strain R1. Both in vivo/in vitro and in vivo/in vivo cross-infections challenges were performed to determine whether prior exposure to either isolate could influence immune responses to subsequent challenge with the other. The results demonstratethat first infection with V. aestuarianus weakened mussel immune defences, thus favoring the subsequent opportunistic behaviour of M. marinus. Chapter 5 the results are reported of a seasonal study on variations in immunocompetence and in the specificity of immune responses of mussels towards three different bacteria: Escherichia coli MG 1655 (not pathogenic to bivalves, used as reference strain), Vibrio aestuarianus 01/32 (pathogenic to oysters but not to mussels), Vibrio tasmaniensis LGP32 (pathogenic to both oysters and mussels). The results support the specificity of mussel immune responses to different bacteria and indicate periods of higher susceptibility to Vibrio infection in winter months, in the spawning season. In the second part (Section II) the research activities related to Task 3.3 on farmed fish are presented (Chapter 6 and 7). A multibiomarker approach was applied to monitor the growth and health status of gilthead seabream Sparus aurata (Chapter 6) and European seabass Dicentrarchus labrax (Chapter 7) reared in an offshore farm in the Ligurian Sea (Lavagna, Genoa, Italy) fed commercial standard diets (Skretting). Monitoring of S. aurata was conducted from the fry to marketable size (15 months), whereas that of D. labrax from a juvenile stage until market size (9 months). Standard morphometric and physiological measurements at the whole organism level indicate good growth performance and health conditions for the examined period for both species. Consistently, biomarker data at the tissue level indicate the absence of oxidative stress conditions or induction of biotransformation processes. In both species, the microbiota composition of anterior intestine samples along growth was evaluated.