Exploring Marine Bacterial Communities with a Focus on Bacteria Attached to Particles

Prokaryotes (Bacteria and Archaea) and phytoplankton have been widely investigated in terms of taxonomy, physiology, metabolism and their role in biogeochemical cycles, however, the relationship with one another is still little known. Phytoplankton can interact with free- living (FL) prokaryotes but can also maintain particle- attached (PA) prokaryotes on their surface, facilitating exchanges in the region surrounding their cells, the phycosphere. In this study, prokaryotic community structures of both PA and FL were determined with the hypothesis that the interaction is not casual but based upon processes linked to the physiology and characteristics of the living particles. In order to obtain a detailed morphological and taxonomical identification of FL and PA prokaryotes, High Throughput 16S rRNA gene Sequencing and Scanning Electron Microscopy were used as main tools. In the first part of the thesis, the prokaryotic community composition was analyzed in diatom cultures, based upon growing evidence that many diatom species rely on mutualistic interactions with their microbiota. Skeletonema marinoi, known to produce PUAs (PolyUnsaturated Aldehydes) and the non PUA-producing Skeletonema tropicum were used to understand if the diatom-attached prokaryotic communities present host-specificity also in relation to the presence or absence of secondary metabolites. In the second and third part of the thesis, the temporal and spatial scales of FL and PA prokaryotes distribution and diversity were investigated in situ to also understand the environmental drivers regulating them. Generally, PA and FL prokaryotes accommodate significantly divergent communities, especially when analyzed at a fine taxonomic level, in relation to the origin and abundance of particles. All the data together provide a clear and comparable assessment of PA and FL prokaryotic diversity, at different sites and under different conditions, making a significant contribution to the field of marine microbial ecology for a better definition of marine microbiome and its potential functional role

Co-occurrence patterns between chaetoceros species and their associated bacteria

Oral presentation 12 th International Phycological Congress, 22–26 March 2021, ChileAutotrophic phytoplankton and heterotrophic bacteria have been investigated in terms of taxonomy, physiology, metabolism and their role in biogeochemical cycles, however the relationship with one another is still little known. This relationship is often governed by microscale interactions played out within the region surrounding phytoplankton cells, called the phycosphere. Phytoplankton can interact with free bacteria but also maintain attached bacteria on their surface. We have analyzed phytoplankton samples from a fixed station in the Gulf of Naples (Italy) sampled weekly from October 2017 to September 2018 for qualitative analysis. Similarly, we have sampled marine bacterial communities with the aim of characterizing them by 16S rDNA sequencing and comparing bacterial communities free-living versus those attached to particles. We then explored interactions between the attached bacteria and the most abundant diatom species using a bipartite network. Chaetoceros spp. was the most abundant diatom species present and, of the 27 species present, only 10 formed statistically significant relationship with specific bacterial taxa. Bipartition network analysis highlighted the presence of 6 different networks, of which three were constituted by single species of Chaetoceros that interact with specific bacterial OTUs, namely, C. vixvisibilis, C. tenuissimus, C. simplex. Instead, the other three networks include multiple species of Chaetoceros with close relationships with specific OTUs but also with other bacterial OTUs common to the other species. Some bacterial phyla co-occurred with all Chaetoceros species while others were diatom-specific (e.g. Cyanobacteria co-occurred only with C. pseudocurvisetus, candidate-phylum-WPS with C. simplex and candidate-phylum-FBP with C. vixvisibilis)ACT has been funded by a SZN-OU fellowshipPeer reviewe

Omics community protocols

The aim of the WP3 “Network Integration and Improvements” is to coordinate and enhance key aspects of integration of European observing technology (and related data flows) for its use in the context of international ocean monitoring activities. One of the dimensions of the integrations is the constitution of thematic networks, that is, networks whose aim is to address specific observational challenges and thus to favor innovation, innovation that will ultimately support the Blue economy. In this context, the specific aim of Task 3.8 is to accelerate the adoption of molecular methods such as genomic, transcriptomic (and related “omics”) approaches, currently used as monitoring tools in human health, to the assessment of the state and change of marine ecosystems. It was designed to favor the increase the capacity to evaluate biological diversity and the organismal metabolic states in different environmental conditions by the development of “augmented observatories”, utilizing state-of-art methodologies in genomic-enabled research at multidisciplinary observatories at well-established marine LTERs, with main focus on a mature oceanographic observatory in Naples, NEREA. In addition, an effort is dedicated to connecting existing observatories that intend to augment their observations with molecular tools. Molecular approaches come with many different options for the protocols (size fractioning, sample collection and storage, sequencing etc). One main challenge in systematically implementing those approaches is thus their standardization across observatories. Based on a survey of existing methods and on a 3-year experience in collecting, sequencing and analyzing molecular data, this deliverable is thus dedicated to present the SOPs implemented and tested at NEREA. The SOPs consider a size fractioning of the biological material to avoid biases toward more abundant, smaller organisms such as bacteria. They cover both the highly stable DNA and the less stable RNA and they are essentially an evolution of the ones developed for the highly successful Tara Oceans Expedition and recently updated for the Expedition Mission Microbiomes, an All-Atlantic expedition organised and executed by the EU AtlantECO project. Importantly, they have only slight variations with respect the ones adopted by the network of genomic observatories EMOBON. Discussions are ongoing with EMOBON to perfectly align the protocols. The SOPs are being disseminated via the main national and international networks

Diversity of Free-Living and Particle-Attached Prokaryotes in a River-Influenced Coastal Area of the Northern Adriatic Sea

13 pages, 7 figures, 1 table, supplementary material https://www.frontiersin.org/articles/10.3389/fmars.2022.912528/full#supplementary-material.-- Data Availability Statement: The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/ - BioProject ID PRJNA834028; accession numbers for single samples are SAMN28028918- SAMN28028941The Northern Adriatic Sea is a key area of the Mediterranean Sea, strongly affected by freshwater inputs, mainly from the Po River, which bring high amounts of nutrients as well as organic and inorganic particles. Free-living and particle-attached prokaryotes were characterized by 16S rRNA gene amplicon sequencing of size-fractionated samples collected during a diatom bloom in this area. The diversity of free-living and particle-attached prokaryotic assemblages was investigated with the aim to understand how the microbial communities are structured in the two fractions and whether specific microbial groups are associated to one lifestyle or the other. The results highlight a diverse prokaryotic community dominated by Proteobacteria, Bacteroidetes, and, remarkably, Firmicutes. Taxa within Firmicutes and Alphaproteobacteria are identified as the main particle-attached indicators by LEfSe, while members of Bacteroidetes and Gammaproteobacteria were representative of the free-living lifestyle, although they were also usually found as particle-attached. Collectively, the results suggest that both the free-living and the particle-attached lifestyles are a complex combination of specialization and adaptation to local conditionsThis is a contribution of RITMARE Flagship Project by Italian MIUR DTA.AD008. AT was supported by a Ph.D. fellowship funded by the Stazione Zoologica Anton Dohrn (Open University-Stazione Zoologica Anton Dohrn Ph.D. Program)With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)Peer reviewe

Phytoplankton Diversity and Co-Dependency in a Stratified Oligotrophic Ecosystem in the South Adriatic Sea

The oligotrophy of the southern Adriatic Sea is characterized by seasonal stratification which enables nutrient supply to the euphotic layer. A set of interdisciplinary methods was used to elucidate the diversity and co-dependency of bacterio- and phytoplankton of the water column during the stratification period of July 2021. A total of 95 taxa were determined by microscopy: 58 diatoms, 27 dinoflagellates, 6 coccolithophores, and 4 other autotrophs, which included Chlorophyceae, Chrysophyceae, and Cryptophytes. Nanophytoplankton abundances were higher in comparison to microphytoplankton. The prokaryotic plankton community as revealed by HTS was dominated by Proteobacteria (41–73%), Bacteroidota (9.5–27%), and cyanobacteria (1–10%), while the eukaryotic plankton community was composed of parasitic Syndiniales (45–80%), Ochrophyta (2–18%), Ciliophora (2–21%), Chlorophytes (2–4%), Haptophytes (1–4%), Bacillariophyta (1–13%), Pelagophyta (0.5–12%) and Chrysophyta (0.5–3%). Flow cytometry analysis has recorded Prochlorococcus and photosynthetic picoeukaryotes as more abundant in deep chlorophyll maximum (DCM), and Synechococcus and heterotrophic bacteria as most abundant in surface and thermocline layers. Surface, thermocline, and DCM layers were distinct considering community diversity, temperature, and nutrient correlations, while extreme nutrient values at the beginning of the investigating period indicated a possible nutrient flux. Nutrient and temperature were recognized as the main environmental drivers of phytoplankton and bacterioplankton community abundance