Spatial and diel variations of bacterioplankton and pico-nanoeukaryote communities and potential biotic interactions during macroalgal blooms.

We investigated spatial heterogeneity and diel variations in bacterioplankton and pico-nanoeukaryote communities, and potential biotic interactions at the extinction stage of the Ulva prolifera bloom in the Jiaozhou Bay, Yellow Sea. It was found that the presence of Ulva canopies significantly promoted the cell abundance of heterotrophic bacteria, raised evenness, and altered the community structure of bacterioplankton. A diel pattern was solely significant for pico-nanoeukaryote community structure. >50 % of variation in the heterotrophic bacterial abundance was accounted for by the ratio of Bacteroidota to Firmicutes, and dissolved organic nitrogen effectively explained the variations in cell abundances of phytoplankton populations. The factors representing biotic interactions frequently contributed substantially more than environmental factors in explaining the variations in diversity and community structure of both bacterioplankton and pico-nanoeukaryotes. There were higher proportions of eukaryotic pathogens compared to other marine systems, suggesting a higher ecological risk associated with the Ulva blooms.

Spatiotemporal assembly and functional composition of planktonic microeukaryotic communities along productivity gradients in a subtropical lake.

Microeukaryotes play crucial roles in the microbial loop of freshwater ecosystems, functioning both as primary producers and bacterivorous consumers. However, understanding the assembly of microeukaryotic communities and their functional composition in freshwater lake ecosystems across diverse environmental gradients remains limited. Here, we utilized amplicon sequencing of 18S rRNA gene and multivariate statistical analyses to examine the spatiotemporal and biogeographical patterns of microeukaryotes in water columns (at depths of 0.5, 5, and 10 m) within a subtropical lake in eastern China, covering a 40 km distance during spring and autumn of 2022. Our results revealed that complex and diverse microeukaryotic communities were dominated by Chlorophyta (mainly Chlorophyceae), Fungi, Alveolata, Stramenopiles, and Cryptophyta lineages. Species richness was higher in autumn than in spring, forming significant hump-shaped relationships with chlorophyll a concentration (Chl-a, an indicator of phytoplankton biomass). Microeukaryotic communities exhibited significant seasonality and distance-decay patterns. By contrast, the effect of vertical depth was negligible. Stochastic processes mainly influenced the assembly of microeukaryotic communities, explaining 63, 67, and 55% of community variation for spring, autumn, and both seasons combined, respectively. Trait-based functional analysis revealed the prevalence of heterotrophic and phototrophic microeukaryotic plankton with a trade-off along N:P ratio, Chl-a, and dissolved oxygen (DO) gradients. Similarly, the mixotrophic proportions were significantly and positively correlated with Chl-a and DO concentrations. Overall, our findings may provide useful insights into the assembly patterns of microeukaryotes in lake ecosystem and how their functions respond to environmental changes.

Starvation alters gut microbiome and mitigates off-flavors in largemouth bass (Micropterus salmoides).

The present study aimed to investigate the response of intestinal microbiota during 3 weeks' starvation of largemouth bass (Micropterus salmoides), an economically important freshwater fish, using 16S rRNA gene amplicon sequencing and PICRUSt2 predictive functional profiling. Overall, the microbiota was mainly represented by Mycoplasma, Pseudomonas, Acinetobacter, and Microbacterium in the initial group. This pattern contrasted with that of Cetobacterium and Aeromonas, which were major representative genera in the starved group. Significant differences in the richness and composition of intestinal microbial community induced by starvation were observed. Notably, earthy-musty off-flavor compounds (geosmin and 2-methylisoborneol) were significantly decreased during starvation, which were significantly correlated with the abundance of certain actinobacterial taxa, namely, Microbacterium and Nocardioides. Additionally, the functional pathways involved in synthesis of off-flavor compounds, protein digestion, fatty acid degradation, and biosynthesis of cofactors greatly decreased with starvation, indicating that microbiota modulated the specific metabolic pathway to adapt to food deprivation. These results emphasize that starvation can modulate diversity, community structure, and functions of the intestinal microbiota and mitigate the off-flavors, which has important implications for strategies to eliminate off-flavor odorants through the application of probiotics to manipulate the gut microbiome and ultimately enhance flesh quality of freshwater fish.

Coupling between Ribotypic and Phenotypic Traits of Protists across Life Cycle Stages and Temperatures.

Relationships between ribotypic and phenotypic traits of protists across life cycle stages remain largely unknown. Herein, we used single cells of two soil and two marine ciliate species to examine phenotypic and ribotypic traits and their relationships across lag, log, plateau, cystic stages and temperatures. We found that Colpoda inflata and Colpoda steinii demonstrated allometric relationships between 18S ribosomal DNA (rDNA) copy number per cell (CNPC), cell volume (CV), and macronuclear volume across all life cycle stages. Integrating previously reported data of Euplotes vannus and Strombidium sulcatum indicated taxon-dependent rDNA CNPC-CV functions. Ciliate and prokaryote data analysis revealed that the rRNA CNPC followed a unified power-law function only if the rRNA-deficient resting cysts were not considered. Hence, a theoretical framework was proposed to estimate the relative quantity of resting cysts in the protistan populations with total cellular rDNA and rRNA copy numbers. Using rDNA CNPC was a better predictor of growth rate at a given temperature than rRNA CNPC and CV, suggesting replication of redundant rDNA operons as a key factor that slows cell division. Single-cell high-throughput sequencing and analysis after correcting sequencing errors revealed multiple rDNA and rRNA variants per cell. Both encystment and temperature affected the number of rDNA and rRNA variants in several cases. The divergence of rDNA and rRNA sequence in a single cell ranged from 1% to 10% depending on species. These findings have important implications for inferring cell-based biological traits (e.g., species richness, abundance and biomass, activity, and community structure) of protists using molecular approaches. IMPORTANCE Based on phenotypic traits, traditional surveys usually characterize organismal richness, abundance, biomass, and growth potential to describe diversity, organization, and function of protistan populations and communities. The rRNA gene (rDNA) and its transcripts have been widely used as molecular markers in ecological studies of protists. Nevertheless, the manner in which these molecules relate to cellular (organismal) and physiological traits remains poorly understood, which could lead to misinterpretations of protistan diversity and ecology. The current research highlights the dynamic nature of cellular rDNA and rRNA contents, which tightly couple with multiple phenotypic traits in ciliated protists. We demonstrate that quantity of resting cysts and maximum growth rate of a population can be theoretically estimated using ribotypic trait-based models. The intraindividual sequence polymorphisms of rDNA and rRNA can be influenced by encystment and temperature, which should be considered when interpreting species-level diversity and community structure of microbial eukaryotes.

Acrylate protects a marine bacterium from grazing by a ciliate predator.

Cleavage of dimethylsulfoniopropionate (DMSP) can deter herbivores in DMSP-producing eukaryotic algae; however, it is unclear whether a parallel defence mechanism operates in marine bacteria. Here we demonstrate that the marine bacterium Puniceibacterium antarcticum SM1211, which does not use DMSP as a carbon source, has a membrane-associated DMSP lyase, DddL. At high concentrations of DMSP, DddL causes an accumulation of acrylate around cells through the degradation of DMSP, which protects against predation by the marine ciliate Uronema marinum. The presence of acrylate can alter the grazing preference of U. marinum to other bacteria in the community, thereby influencing community structure.

Taxonomic Diversity of Pico-/Nanoeukaryotes Is Related to Dissolved Oxygen and Productivity, but Functional Composition Is Shaped by Limiting Nutrients in Eutrophic Coastal Oceans.

Pico-/nanoeukaryotes (P/NEs) comprise both primary producers and bacterial predators, playing important biogeochemical and ecological roles in the marine microbial loop. Besides the difference in size, these small-sized fractions can be distinguished from microplankton by certain functional and ecological traits. Nevertheless, little information is available regarding patterns of their taxonomic and functional diversity and community composition along environmental gradients in coastal marine ecosystems. In this study, we applied high-throughput sequencing of 18S rRNA gene to assess the taxonomic species richness and community composition of P/NEs in surface waters of Bohai Sea and North Yellow Sea, northern China spanning a 600-km distance during summer and winter of 2011. The richness of operational taxonomic units (OTUs) formed a U-shaped relationship with concentration of chlorophyll a (Chl-a, a proxy of primary productivity), but a stronger, negative relationship with concentration of dissolved oxygen (DO). These two factors also significantly co-varied with the OTU-based community composition of P/NEs. The effect of geographic distance on community composition of P/NEs was negligible. Among the three functional groups defined by trophic traits, heterotrophs had the highest OTU richness, which exhibited a U-shaped relationship with both DO and Chl-a. The community of P/NEs was dominated by heterotrophs and mixotrophs in terms of read numbers, which showed a trade-off along the gradient of phosphate, but no significant changes along DO and Chl-a gradients, indicating functional redundancy. Similarly, the proportion of phototrophs was significantly and positively correlated with the concentration of silicate. Our results indicate that taxonomic and functional composition of P/NEs are decoupled on a regional scale, and limiting nutrients are important factors in modulating functional composition of these microorganisms in the studied area. These findings contribute toward gaining a better understanding of how diversity of small eukaryotes and their functions are structured in coastal oceans and the effect of environmental changes on the structuring process.

Comparative Transcriptomics Reveals Distinct Gene Expressions of a Model Ciliated Protozoa Feeding on Bacteria-Free Medium, Digestible, and Digestion-Resistant Bacteria.

Bacterivory is an important ecological function of protists in natural ecosystems. However, there are diverse bacterial species resistant to protistan digestion, which reduces the carbon flow to higher trophic levels. So far, a molecular biological view of metabolic processes in heterotrophic protists during predation of bacterial preys of different digestibility is still lacking. In this study, we investigated the growth performance a ciliated protozoan Tetrahymena thermophila cultivated in a bacteria-free Super Proteose Peptone (SPP) medium (control), and in the media mixed with either a digestion-resistant bacterial species (DRB) or a digestible strain of E. coli (ECO). We found the protist population grew fastest in the SPP and slowest in the DRB treatment. Fluorescence in situ hybridization confirmed that there were indeed non-digested, viable bacteria in the ciliate cells fed with DRB, but none in other treatments. Comparative analysis of RNA-seq data showed that, relative to the control, 637 and 511 genes in T. thermophila were significantly and differentially expressed in the DRB and ECO treatments, respectively. The protistan expression of lysosomal proteases (especially papain-like cysteine proteinases), GH18 chitinases, and an isocitrate lyase were upregulated in both bacterial treatments. The genes encoding protease, glycosidase and involving glycolysis, TCA and glyoxylate cycles of carbon metabolic processes were higher expressed in the DRB treatment when compared with the ECO. Nevertheless, the genes for glutathione metabolism were more upregulated in the control than those in both bacterial treatments, regardless of the digestibility of the bacteria. The results of this study indicate that not only bacterial food but also digestibility of bacterial taxa modulate multiple metabolic processes in heterotrophic protists, which contribute to a better understanding of protistan bacterivory and bacteria-protists interactions on a molecular basis.

Morphology and Phylogeny of the Soil Ciliate Metopus yantaiensis n. sp. (Ciliophora, Metopida), with Identification of the Intracellular Bacteria.

The morphology and infraciliature of a new ciliate, Metopus yantaiensis n. sp., discovered in coastal soil of northern China, were investigated. It is distinguished from its congeners by a combination of the following features: nuclear apparatus situated in the preoral dome; 18-21 somatic ciliary rows, of which three extend onto the preoral dome (dome kineties); three to five distinctly elongated caudal cilia, and 21-29 adoral polykinetids. The 18S rRNA genes of this new species and two congeners, Metopus contortus and Metopus hasei, were sequenced and phylogenetically analyzed. The new species is more closely related to M. hasei and the clevelandellids than to other congeners; both the genus Metopus and the order Metopida are not monophyletic. In addition, the digestion-resistant bacteria in the cytoplasm of M. yantaiensis were identified, using a 16S rRNA gene clone library, sequencing, and fluorescence in situ hybridization. The detected intracellular bacteria are affiliated with Sphingomonadales, Rhizobiales, Rickettsiales (Alphaproteobacteria), Pseudomonas (Gammaproteobacteria), Rhodocyclales (Betaproteobacteria), Clostridiales (Firmicutes), and Flavobacteriales (Bacteroidetes).

Protist-Bacteria Associations: Gammaproteobacteria and Alphaproteobacteria Are Prevalent as Digestion-Resistant Bacteria in Ciliated Protozoa.

Protistan bacterivory, a microbial process involving ingestion and digestion, is ecologically important in the microbial loop in aquatic and terrestrial ecosystems. While bacterial resistance to protistan ingestion has been relatively well understood, little is known about protistan digestion in which some ingested bacteria could not be digested in cells of major protistan grazers in the natural environment. Here we report the phylogenetic identities of digestion-resistant bacteria (DRB) that could survive starvation and form relatively stable associations with 11 marine and one freshwater ciliate species. Using clone library and sequencing of 16S rRNA genes, we found that the protistan predators could host a high diversity of DRB, most of which represented novel bacterial taxa that have not been cultivated. The localization inside host cells, quantity, and viability of these bacteria were checked using fluorescence in situ hybridization. The DRB were affiliated with Actinobacteria, Bacteroidetes, Firmicutes, Parcubacteria (OD1), Planctomycetes, and Proteobacteria, with Gammaproteobacteria and Alphaproteobacteria being the most frequently occurring classes. The dominance of Gamma- and Alphaproteobacteria corresponds well to a previous study of Global Ocean Sampling metagenomic data showing the widespread types of bacterial type VI and IV secretion systems (T6SS and T4SS) in these two taxa, suggesting a putatively significant role of secretion systems in promoting marine protist-bacteria associations. In the DRB assemblages, opportunistic bacteria such as Alteromonadaceae, Pseudoalteromonadaceae, and Vibrionaceae often presented with high proportions, indicating these bacteria could evade protistan grazing thus persist and accumulate in the community, which, however, contrasts with their well-known rarity in nature. This begs the question whether viral lysis is significant in killing these indigestible bacteria in microbial communities. Taken together, our study on the identity of DRB sheds new light on microbial interactions and generates further hypotheses including the potential importance of bacterial protein secretion systems in structuring bacterial community composition and functioning of "microbial black box" in aquatic environments.