Biogeography and its impact on benthic heterotrophic protists – Investigations from the littoral to the deep sea

Biogeography is the study of the distribution of biodiversity and the processes resulting in spatial patterns. Knowledge on biogeography is crucial for the understanding of ecosystem functions and evolutionary processes. But, if biogeographical concepts, established for macroorganisms, also apply for microbial communities is far from being clarified. Protists (unicellular eukaryotes) are the most common and most diverse eukaryotes on Earth and play a key role in the microbial food web by transferring carbon to higher trophic levels. However, their diversity and distribution in marine sediments, especially in the deep sea, is yet not fully understood. The overall aim of this study was to extend the knowledge on biogeographical distribution patterns of protist communities at the ocean floor considering different spatial scales to understand ecological and evolutionary processes within this vast environment. By combining the molecular technique of amplicon sequencing with live-countings, direct microscopical observations and cultivation-based approaches, benthic protist communities along depth transects around multiple islands and seamounts of the Azores archipelago were investigated. Strong differences between communities from different depths and islands were found, indicating a separation of communities on islands shelfs/seamounts from communities inhabiting the surrounding deep-sea areas, favouring speciation processes on islands/seamounts. The deep-sea areas surrounding islands and seamounts are complex environments, characterised by large geological features and diverse microhabitats, possibly enhancing the evolution of a diverse protist community, specially adapted to the complex and harsh prevailing conditions. Within this study, the composition and distribution of deep-sea protist communities in bathyal, abyssal and hadal depths were analysed. Amplicon sequencing of the V9 18S rDNA of sediment samples from three different abyssal regions in the North Atlantic Ocean and the Caribbean Sea, revealed high variations in the structure of protist communities at large spatial scales. In addition, spatial patterns at local scales were found by comparing community compositions of sediment samples taken ~1 m apart from each other. The OTU (operational taxonomic units) richness decreased with sediment depth which correlated with decreasing TOM (total organic matter) contents, when looking at a vertical scale in different sediment layers of the same sediment core. The only exception was one station where sedimented macroalgae (Sargassum) were found on the deep-sea floor, possibly influencing the microbial communities directly or indirectly. A global study, comparing 11 regions in the Atlantic and Pacific Ocean, showed that different ocean basins harbour unique protist communities with high rates of endemism, being distinct from surface water communities. A high genetic novelty was found within the deep-sea samples, indicating a specific deep-sea nanofauna, but it also underlines the need to extend common reference databases using cultivated protist strains. Within this study, two protist groups, with clonal cultures from surface waters and the deep sea, were investigated more in detail to examine their biogeographical distribution and ecological adaptations. Fourteen strains of Percolomonadida were isolated and cultivated, including two new families, four new genera and ten newly described species. Phylogenetic analyses indicated a separation by ecological traits of the marine families Lulaidae and Barbeliidae from the Percolomonadidae with representatives from different meso- and hypersaline waters. The isolation and cultivation of 39 strains of Cafeteria-like flagellates led to the reorganization of the Cafeteriaceae with nine newly described species being added to the two already known species. Within the Percolomonadida as well as the Cafeteriaceae, single species seemed to be globally distributed in marine surface waters and the deep sea and were able to survive varying environmental conditions (salinity and hydrostatic pressure). However, most species seemed to be locally restricted in their distribution. Overall, the combination of various different methods enabled a broad overview on the diversity and distribution of benthic protist communities from the sublittoral down to the deep sea and showed that different islands of the Azores harbour distinct protist communities. Spatial distribution patterns were observed at local, regional and global scales in the deep sea, indicating that protist communities are shaped by the heterogeneity of the deep-sea floor. Detailed analyses of single taxonomic groups gave new insights into their biogeographical distribution and adaptations to different environmental conditions. The present thesis underlines the complex biogeographical distribution patterns of protist communities at different spatial scales, probably shaped by heterogenous environments with changing, extreme conditions, which require specific ecological adaptations

Amplicon sequence variant table of benthic heterotrophic protists of the southern Baltic Sea sampled in 2020-2022

We aimed to explore the community compostion of benthic heterotrophic protists in three different regions of the southern Baltic Sea - Fehmarnbelt, Oderbank and Roennebank. Sediment samples where collected with a multicorer system and sliced into layered depth profile during three cruises in 2020, 2021 and 2022 with research vessels Elisabeth Mann Borgese (2020, EMB238 and 2021, EMB267) and Alkor (2022, AL570). We performed a paired-end NovaSeq sequencing (2 × 150 bp) run of the amplified V9 region of the 18S rDNA. For subsequent quality measures during data analysis, we created an in vitro community, called a "mock community", comprising DNA of nine different protist cultures, adding this mixture to each individual sequencing run. After sequencing, the raw reads were demultiplexed and the barcode and primer sequences were clipped using cutadapt (Martin 2011). The data was further processed using the the dada2 package in R (Callahan et al. 2016). For taxonomic assignment we used the PR2 database (Protist Ribosomal Reference database, Guillou et al. 2012, https://pr2-database.org/ ) updated with 150 sequences obtained from our own collection using usearch_global (v2.18.0, Rognes et al. 2016). We discarded all Metazoa, fungi, autotrophic protists (determined on the basis of taxonomic assignment) and retained only heterotrophic protists' amplicon sequence variants (ASVs) with a pairwise identity of >80% to a reference sequence. For the main dataset of samples, we then chose individual minimum thresholds per sample according to the accompanying mock community on the respective sequencing lane. For calculation of these thresholds, we used the proportion of the lowest read number of an ASV in the mock community data set that could be assigned to the cultured species

Environmental data related to amplicon sequencing of benthic heterotrophic protists of the southern Baltic Sea, in 2020-2022

We aimed to explore the community compostion of benthic heterotrophic protists in three different regions of the southern Baltic Sea - Fehmarnbelt, Oderbank and Roennebank. Sediment samples where collected with a multicorer system and sliced into layered depth profile during three cruises in 2020, 2021 and 2022 with research vessels Elisabeth Mann Borgese (2020, EMB238 and 2021, EMB267) and Alkor (2022, AL570). We performed a paired-end NovaSeq sequencing (2 × 150 bp) run of the amplified V9 region of the 18S rDNA. This dataset includes the environmental data related to the sequencing study including sample IDs, region, sediment depth, station data, salinity and median grain size

The Portal Vertex of KSHV Promotes Docking of Capsids at the Nuclear Pores

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-related herpesvirus. Like other herpesviruses, the KSHV icosahedral capsid includes a portal vertex, composed of 12 protein subunits encoded by open reading frame (ORF) 43, which enables packaging and release of the viral genome into the nucleus through the nuclear pore complex (NPC). Capsid vertex-specific component (CVSC) tegument proteins, which directly mediate docking at the NPCs, are organized on the capsid vertices and are enriched on the portal vertex. Whether and how the portal vertex is selected for docking at the NPC is unknown. Here, we investigated the docking of incoming ORF43-null KSHV capsids at the NPCs, and describe a significantly lower fraction of capsids attached to the nuclear envelope compared to wild-type (WT) capsids. Like WT capsids, nuclear envelope-associated ORF43-null capsids co-localized with different nucleoporins (Nups) and did not detach upon salt treatment. Inhibition of nuclear export did not alter WT capsid docking. As ORF43-null capsids exhibit lower extent of association with the NPCs, we conclude that although not essential, the portal has a role in mediating the interaction of the CVSC proteins with Nups, and suggest a model whereby WT capsids can dock at the nuclear envelope through a non-portal penton vertex, resulting in an infection ‘dead end’