Dynamics of microbial community during faecal egestion of clams (Ruditapes philippinarum) under controlled conditions

ABSTRACT Bivalve molluscs belong to the phylum Mollusca, and they are aquatic invertebrate. Bivalve molluscs are a food product of a great economic importance and highly valued by consumers, but one of the main characteristics of shellfish is that they are filtered feeders, and they can accumulate toxic substances like chemicals, microorganisms and contaminants present in the seawater. Among these microorganisms, are some that may be pathogenic to humans, for this reason, the bivalve molluscs can only be put on the market after depuration for removing all dangerous microorganisms. One of the most important species of bivalve molluscs is Manila clams (Ruditapes philippinarum). This study is focused on this species and the aim of the project is to study the dynamics of microbial community during faecal egestion in two different times, after 3 and 5 hours from sample collection. The "microbiota", or microbial populations, is crucial for clam growth, health, and defence against pathogenic diseases. By combining cultural and non-cultural methodologies, it was possible to define various images of the bacteria's release into the jar, analyzing faecal egestion quantities. Through a culture analysis, it was possible to determine a quantitative estimate of marine bacteria in the samples and faecal egestion, grown on Marine agar, and Vibrio grown on TCBS. From the obtained results, it was determined that the water has a lower number of microorganisms compared to the homogenate and egestion samples, which exhibited a large quantity of bacteria, particularly in the egestion 5h into the first day and the homogenate of the second. Comparing the results of microbiology of 2022 and 2023 there was significant increase in microbial activity in 2023 versus the previous year. From the gills, glands, homogenate, water and egestion, the DNA of the microbial community was extracted. Information on bacterial biodiversity and bacterial genera present in the samples was obtained from the 16S sequences. The microbial community was analysed using 16S sequencing using Next Generation Sequencing. This technology has revolutionized microbial ecology studies because with these new methodologies it is possible to define the main genus of bacteria present in the microbiota of clams. This approach led to the identification of the predominant 20 genus presents in the matrices, the three principals are: Endozoicomonas, Mycoplasma and Vibrio. Further analysis have indicated a high number of taxa in the homogenate and egestion and also in the organ. In contrast, the water sample exhibited a lower value compared to the other. Additionally, beta diversity analysis was conducted to assess the diversity between the samples and has been investigated also with the PERMANOVA analysis. The results revealed that the pairwise comparison showed significant differences between egestion and homogenate. From the results obtained for the gills and glands shown a statically difference in the comparison of the two different organs but the difference is not relevant between glands pre and 5 hours and gill pre and 5 hours.