Seasonal fluctuations of hemolymph microbiota and immune parameters in Mytilus galloprovincialis farmed at La Spezia, Italy

Marine bivalves include economically highly important aquacultured species worldwide (oysters, mussels, clams, scallops). Although one third of production in Europe is represented by mussels (Mytilus edulis, M. galloprovincialis) a significant decline has been observed in the last two decades, whose causes are likely to be multifactorial, from the presence of pathogens to climate change, often exacerbated by local conditions, leading to mortality outbreaks. Knowledge of the composition of the microbial communities, together with their relationship with the host, represents a primary target for understanding bivalve health in different environmental conditions. The microbiome is strongly influenced by both local environmental factors and endogenous factors, such as the host immune status. However, information on the seasonal fluctuations of microbial communities in mussels, together with immune parameters, is still lacking. In the present work, M. galloprovincialis cultivated in north-western Italy (La Spezia) were sampled from February to November 2020 and hemolymph microbiome was evaluated using 16S rRNA gene, together with a set of immune biomarkers in hemocytes (lysosomal membrane stability-LMS, phagocytic activity, ROS production) and soluble lysozyme activity, and parameters of general health conditions (gametogenesis, survival in air). The results show that M. galloprovincialis hemolymph microbiota was characterized by the constant presence of 8 bacterial genera (Vibrio, Pseudoalteromonas, Tenacibaculum, Colwellia, Shewanella, Amphritea, and 2 from the Arcobacteracea family, Halarcobacter and Arcobacter-like). Seasonal changes in their relative abundance were observed, showing a distinct pattern in winter (February-November) and summer (July, September). Water temperature contributed to shaping the microbiota composition, in particular with regards to the abundance of Vibrios, Psychrilobacter and Photobacterium, Halarcobacter and Arcobacter-like, that was favored by higher temperatures. Seasonal variations of LMS and phagocytosis were also observed, indicating a reduced immunocompetence in winter, concomitant with general stressful conditions associated with spawning. In contrast, the main fluctuations in extracellular immune defences (ROS and lysozyme) were apparently independent of temperature or gametogenic stage. Overall, the results confirm the robustness of mussel immune system, at the same time underlying the potential susceptibility to disease at certain times of the year to additional stressful conditions (i.e. pathogen challenge, pollutant exposure, or further changes in water chemico-physical parameters). These data provide background information useful for monitoring local mussel farming and predicting potential impacts of changing environmental conditions.