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Arbuscular mycorrhizal (AM) fungi lack efficient exoenzymes to access organic nutrients directly. Nevertheless, the fungi often obtain and further channel to their host plants a significant share of nitrogen (N) and/or phosphorus from such resources, presumably via cooperation with other soil microorganisms. Because it is challenging to disentangle individual microbial players and processes in complex soil, we took a synthetic approach here to study (15)N-labelled chitin (an organic N source) recycling via microbial loop in AM fungal hyphosphere. To this end, we employed a compartmented in vitro cultivation system and monoxenic culture of Rhizophagus irregularis associated with Cichorium intybus roots, various soil bacteria, and the protist Polysphondylium pallidum. We showed that upon presence of Paenibacillus sp. in its hyphosphere, the AM fungus (and associated plant roots) obtained several-fold larger quantities of N from the chitin than it did with any other bacteria, whether chitinolytic or not. Moreover, we demonstrated that adding P. pallidum to the hyphosphere with Paenibacillus sp. further increased by at least 65% the gain of N from the chitin by the AM fungus compared to the hyphosphere without protists. We thus directly demonstrate microbial interplay possibly involved in efficient organic N utilisation by AM fungal hyphae.

The role of mixoplanktonic algae is increasingly recognised as an important link between primary production and microbial loop processes, particularly in dystrophic systems where staining can both limit light and nutrient availability and increase bacterial productivity. We hypothesised that these three factors would affect the relative abundance of mixotrophs (RAmixotrophs), with increased light and nutrient availability decreasing RAmixotrophs and increased bacterial availability increasing RAmixotrophs. We employed a fully factorial design within near-shore mesocosms in a highly stained lake, with light intensity, bacterial abundance, and phosphorus concentration as treatment variables. Over the initial 3 days of the experiment, when changes were greatest, we found no significant three-way interaction effect among light, phosphorus and nutrients on the RAmixotrophs. We did find significant two-way interactions between phosphorus and light, and phosphorus and bacteria. RAmixotrophs was decreased by the addition of phosphorus whether light was added or not, but light only significantly deceased RAmixotrophs when phosphorus was not added. Phosphorus concentration was also found to decrease RAmixotrophs whether bacterial food was supplemented or not, but bacteria increased RAmixotrophs only in mesocosms without phosphorus additions. Collectively, our results indicated that phosphorus concentrations may be the primary driver of mixoplanktonic phytoplankton dynamics in dystrophic systems.

Soil microbial communities, key players of many crucial ecosystem functions, are susceptible to environmental disturbances, which might cause the loss of microbial diversity and functions. However, few ecological concepts and practices have been developed for rescuing stressed soil microbial communities. Here, we manipulated an experiment with or without arbuscular mycorrhizal fungi (AMF) inoculation and at three levels (one, three and six species) of plant diversity to disentangle how the AMF and vegetation rescue soil nitrogen (N) -cycling microbial loop from simulated degraded soil ecosystem. Our results showed that AMF inoculation improved the restoration of soil N-cycling microbial communities. This improved restoration was related to the role of AMF in enhancing interactions within the N-cycling microbial loop. Furthermore, increased plant diversity strengthened the role of AMF in rescuing N-cycling microbial communities. Our findings provide novel insights into the roles of AMF and plant diversity in facilitating the rescue of microbial communities in degraded terrestrial ecosystems.

Background Cooling towers are a major source of large community-associated outbreaks of Legionnaires’ disease, a severe pneumonia. This disease is contracted when inhaling aerosols that are contaminated with bacteria from the genus Legionella, most importantly Legionella pneumophila. How cooling towers support the growth of this bacterium is still not well understood. As Legionella species are intracellular parasites of protozoa, it is assumed that protozoan community in cooling towers play an important role in Legionella ecology and outbreaks. However, the exact mechanism of how the eukaryotic community contributes to Legionella ecology is still unclear. Therefore, we used 18S rRNA gene amplicon sequencing to characterize the eukaryotic communities of 18 different cooling towers. The data from the eukaryotic community was then analysed with the bacterial community of the same towers in order to understand how each community could affect Legionella spp. ecology in cooling towers. Results We identified several microbial groups in the cooling tower ecosystem associated with Legionella spp. that suggest the presence of a microbial loop in these systems. Dissolved organic carbon was shown to be a major factor in shaping the eukaryotic community and may be an important factor for Legionella ecology. Network analysis, based on co-occurrence, revealed that Legionella was correlated with a number of different organisms. Out of these, the bacterial genus Brevundimonas and the ciliate class Oligohymenophorea were shown, through in vitro experiments, to stimulate the growth of L. pneumophila through direct and indirect mechanisms. Conclusion Our results suggest that Legionella ecology depends on the host community, including ciliates and on several groups of organisms that contribute to its survival and growth in the cooling tower ecosystem. These findings further support the idea that some cooling tower microbiomes may promote the survival and growth of Legionella better than others.

Planktonic ciliates have been recognized as major consumers of nano- and picoplankton in pelagic ecosystems, playing pivotal roles in the transfer of matter and energy in the microbial loop. However, due to the difficulties in identification, the species composition of ciliate assemblages, especially for the small, fragile, and naked species that usually dominate the ciliate communities in the oceanic waters, remains largely unknown. In the present study, 22 stations along the transect from Shenzhen (China) to Pohnpei (Micronesia) were sampled for the enumeration of picoplankton and nanoflagellates. In addition, pigment analysis of major phytoplankton groups along with the measurements of environmental variables including temperature, salinity, and nutrients were also carried out. Ciliates were identified at species level using quantitative protargol stain to reveal the species composition and their distribution patterns from off-shore to open ocean. Ciliate abundance was positively correlated with phosphate, silicate, and pico-sized pigmented eukaryotes (PPEs), whereas the biomass was closely related with PPEs, heterotrophic nanoflagellates, and chlorophytes. The combination of silicate and pigmented nanoflagellates was identified as the major factor driving the ciliate community composition. The close relationship between silicate and ciliate abundance and community structure needs further validation based on more data collected from oceanic waters. Our study showed the necessity of using techniques that can reveal the community composition at higher taxonomic resolutions in future studies on ciliates. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42995-020-00075-7) contains supplementary material, which is available to authorized users.

The effects of diatom abundance and biogenic silica (BSi) deposition on the population growth of tintinnid ciliates were investigated using incubations of sub-surface waters (10 m depth) of Suruga Bay, Japan in April and November 2018. Seawater for incubation was pre-filtered using a 200-µm mesh to remove mesozooplankton grazers. Initial concentrations of dissolved inorganic nitrogen were similar in both seasons, but silicic acid and phosphate were higher in April than in November. This allowed us to observe the growth of planktonic communities under varied silicic acid availability and explore the effects of BSi accumulation and food availability on the growth of tintinnids. Phytoplankton blooms dominated by diatoms formed in both, April and November incubations, but their biomass was higher in April than in November, demonstrating the effects of nutrients, particularly silicic acid, on diatom growth. The BSi concentration increased in both incubations but was higher in April than in November, confirming the effects of diatom abundance and size on BSi deposition. Tintinnid biomass also increased in both incubations but was higher in April than in November, showing that the biomass of tintinnid was affected by the BSi concentration. Species composition of diatoms affected the species composition of tintinnids by providing selected material for the construction of their lorica. Moreover, the results showed that the food supply mainly provided by pico-nanoflagellates via microbial loop and detritus may limit tintinnids’ population growth in natural planktonic ecosystems and this demonstrates tintinnids’ role as top predators in the microbial loop

Benthic primary producers in coastal ecosystems provide important habitat for marine organisms through the provision of complex 3D habitat. Primary producers produce organic matter, while simultaneously producing reactive oxygen species, including hydrogen peroxide (H2O2), a driver of oxidative stress. Through their high biomass, productivity and effect on local hydrodynamics, benthic primary producers can potentially increase H2O2 concentrations surrounding the biogenic structures they form. The aim of this study was to identify the potential role of H2O2 produced by benthic primary producers as an external stressor in coastal ecosystems. This was achieved by measuring H2O2 concentrations within sea lettuce blooms (Ulva sp.), giant kelp forests (Macrocystis pyrifera), and seagrass meadows (Zostera muelleri); quantifying H2O2 production rates of these species; and testing heterotrophic bacterial response to relevant H2O2 concentrations. Ulva sp. produced five times more H2O2 than other species. At in situ concentrations, H2O2 inhibited bacterial production and carbon flow through the microbial loop by 75%. This study reveals H2O2 as an additional stressor in bloom-forming Ulva sp. with higher H2O2 production compared to the ecosystem engineers M. pyrifera and Z. muelleri. H2O2 production by benthic primary producers can affect carbon flow through the microbial loop, with the potential to propagate a stress signal up the food web.

This work aims to outline the dynamics of trophic links between the three main microbial components (bacteria, nanoflagellates, and ciliates) of the Farasan Archipelago in order to establish a baseline for future research in this area. The Farasan Archipelago lies along the southwestern coast of the Saudi Arabia, southern Red Sea between 16°20′–17°10′N and 41°30′–42°30′E and had been declared as marine and terrestrial reserve by the year 1996. Three different sites were chosen for this study, with each site visited bimonthly for 18 months from September 2016 to February 2018. Bacteria, nanoflagellates and ciliates were enumerated in order to explore the complex interactions between the main microbial categories in sea waters of the Farasan Archipelago. High abundances were recorded during the present study for bacteria (8.7 × 106 bacteria ml−1), nanoflagellates (3.7 × 104 TNAN ml−1) and ciliates (40.4 ciliates ml−1). The paper discusses the various potential pathways controlling the complex interactions between these microbial groups in this part of the southern Red Sea. It is concluded that a linear trophic chain consisting of bacteria; heterotrophic nanoflagellates; filter feeding ciliates is a major route by which the production of bacteria is transferred to the higher consuming levels, thereby confirming the high importance of t bottom-up control (food supply), alongside top-down control (predation) in regulating bacterial abundances in the Farasan Archipelago. During the present investigation, each nanoflagellate ingested between 11 and 87 bacteria in one hour, while each ciliate consumed between 20 and 185 nanoflagellates every hour. These calculated grazing rates of protistan eukaryotes confirmed the role of heterotrophic nanoflagellates as the main consumers of bacteria, and the role of ciliates as the major control for the heterotrophic nanoflagellate population dynamics, and thus the top predators within the microbial plankton assemblage in the Farasan Archipelago. Keywords: Heterotrophic bacteria, Interactions, Microbial loop, Protistan grazers

Lake Cadagno, a permanently stratified high-alpine lake with a persistent microbial bloom in its anoxic chemocline, has long been considered a model for the low-oxygen, high-sulfide Proterozoic ocean where early microbial life gave rise to Earth’s oxygenated atmosphere. Although the lake has been studied for over 25 years, the absence of concerted study of the bacteria, phytoplankton, and viruses, together with primary and secondary production, has hindered a comprehensive understanding of its microbial food web. Here, the identities, abundances, and productivity of microbes were evaluated in the context of Lake Cadagno biogeochemistry. Photo-synthetic pigments and chloroplast 16S rRNA gene phylogenies suggested high abundances of eukaryotic phytoplankton, primarilyChlorophyta, through the water column. Of these, a close relative ofAnkyra judayi, a high-alpine adapted chlorophyte, peaked with oxygen in the mixolimnion, whileClosteriopsis-related chlorophytes peaked in the chemocline and monimolimnion. Anoxygenic phototrophic sulfur bacteria, Chromatium, dominated the chemocline along withLentimicrobium, a newly observed genus of known fermenters. Secondary production peaked in the chemocline, suggesting anoxygenic primary producers depended on heterotrophic nutrient remineralization. Virus-to-microbe ratios spanned an order of magnitude, peaking with high phytoplankton abundances and at a minimum at the peak of Chromatium, dynamic trends that suggest viruses may play a role in the modulation of oxygenic and anoxygenic photo- and chemosynthesis in Lake Cadagno. Through the combined analysis of bacterial, eukaryotic, viral, and biogeochemical dynamics of Lake Cadagno, this study provides a new perspective on the biological and geochemical connections that comprised the food webs of the Proterozoic ocean.IMPORTANCEAs a window to the past, the study offers insights into the role of microbial guilds of Proterozoic ocean chemoclines in the production and recycling of organic matter of sulfur- and ammonia-containing ancient oceans. The new observations described here suggest that eukaryotic algae were persistent in the low oxygen upper-chemocline in association with purple and green sulfur bacteria in the lower half of the chemocline. Further, this study provides the first insights into Lake Cadagno viral ecology. High viral abundances suggested viruses may be essential components of the chemocline where their activity may result in the release and recycling of organic matter. The framework developed in this study through the integration of diverse geochemical and biological data types lays the foundation for future studies to quantitatively resolve the processes performed by discrete populations comprising the microbial loop in this early anoxic ocean analogue.

Significance Marine dissolved organic matter, which originates from phytoplankton, holds as much carbon as Earth’s atmosphere; yet, the biological processes governing its fate are primarily studied under idealized laboratory conditions or through indirect measures such as genome sequencing. In this work, we used isotope labeling to directly quantify uptake of complex carbon pools from the two primary sources of marine organic carbon (diatoms and cyanobacteria) by a natural microbial community. Our data show that carbon pools are partitioned into distinct microbial lineages whose physiological properties and resource acquisition strategies match the chemical nature of their preferred substrates. Our results provide ecological and functional insights into the patterns of microbial community structure changes that occur during marine phytoplankton blooms., Blooms of marine phytoplankton fix complex pools of dissolved organic matter (DOM) that are thought to be partitioned among hundreds of heterotrophic microbes at the base of the food web. While the relationship between microbial consumers and phytoplankton DOM is a key component of marine carbon cycling, microbial loop metabolism is largely understood from model organisms and substrates. Here, we took an untargeted approach to measure and analyze partitioning of four distinct phytoplankton-derived DOM pools among heterotrophic populations in a natural microbial community using a combination of ecogenomics, stable isotope probing (SIP), and proteomics. Each 13C-labeled exudate or lysate from a diatom or a picocyanobacterium was preferentially assimilated by different heterotrophic taxa with specialized metabolic and physiological adaptations. Bacteroidetes populations, with their unique high-molecular-weight transporters, were superior competitors for DOM derived from diatom cell lysis, rapidly increasing growth rates and ribosomal protein expression to produce new relatively high C:N biomass. Proteobacteria responses varied, with relatively low levels of assimilation by Gammaproteobacteria populations, while copiotrophic Alphaproteobacteria such as the Roseobacter clade, with their diverse array of ABC- and TRAP-type transporters to scavenge monomers and nitrogen-rich metabolites, accounted for nearly all cyanobacteria exudate assimilation and produced new relatively low C:N biomass. Carbon assimilation rates calculated from SIP data show that exudate and lysate from two common marine phytoplankton are being used by taxonomically distinct sets of heterotrophic populations with unique metabolic adaptations, providing a deeper mechanistic understanding of consumer succession and carbon use during marine bloom events.*

A mass-balance model simulating a biotic energy flow in the plankton community with the participation of microbial organisms has been designed for a lake-type ecosystem. The input abiotic parameters of the model are latitude, mean depth, water color, and total phosphorus content in water. The model was constructed on the basis of the data published for the Rybinsk Reservoir and Mongolian lakes. An analysis of the model indicated that the addition of the microbial loop to the traditional linear trophic chain resulted in a twofold decrease in the biomass and production of metazoan plankton and planktivorous fish. The value of the final ecosystem production (fish production) is suggested to be closely associated with the degree of development of the microbial loop during the growing season.

Bivalves represent one of the most important components of freshwater zoobenthos, and their shells provide a substrate for many organisms to create epibiotic communities of predominantly facultative nature. Thus far, information regarding colonization of Unio tumidus by microorganism assemblages has been almost absent. Moreover, data on the functioning of trophic networks created by epibionts are also unavailable. Therefore, the present study aimed to examine the assemblage of epibiotic microorganisms inhabiting live and dead bivalves with regard to the physicochemical properties of the habitat and determine whether stable isotope levels in primary producers and consumers colonizing bivalve shells exhibit significant seasonal variability. The substrate characteristic and physicochemical properties of water – primarily biogenic compounds and total organic carbon clearly modified the taxonomic composition of microorganisms and the function of the trophic network. This was reflected by the increased diversity of microorganisms (phycoflora, ciliates and rotifers) on live bivalves and the increase in their abundance on dead bivalves. The level of stable isotopes exhibited clear seasonal variability in individual components of the epibiotic trophic network, whereas food preferences of the potential consumers (protozoans and rotifers) depended significantly on the availability and abundance of food.

An intricate set of interactions characterizes marine ecosystems. One of the most important is represented by the so-called microbial loop, which includes the exchange of dissolved organic matter (DOM) from phototrophic organisms to heterotrophic bacteria. Here, it can be used as the major carbon and energy source. Arguably, this interaction is one of the foundations of the entire ocean food-web. Carbon fixed by phytoplankton can be redirected to bacterial cells in two main ways; either i) bacteria feed on dead (eventually lysed) phytoplankton cells or ii) DOM is actively released by phytoplankton cells (a widespread process that may result in up to 50% of the fixed carbon leaving the cell). In this work, we have set up a co-culture of the model diatom Phaeodactylum tricornutum and the model chemoheterotrophic bacterium Pseudoalteromonas haloplanktis TAC125 and used this system to study the interactions between these two representatives of the microbial loop. We show that the bacterium can indeed thrive on diatom-derived carbon and that this growth can be sustained by both diatom dead cells and diatom-released compounds. These observations were formalized in a network of putative interactions between P. tricornutum and P. haloplanktis and implemented in a mathematical model that reproduces the observed co-culture dynamics, suggesting that our hypotheses on the interactions occurring in this two-player system can accurately explain the experimental data.

Meromictic Lake Cadagno, an ancient ocean analogue, is known for its permanent stratification and persistent anoxygenic microbial bloom within the chemocline. Although the anaerobic microbial ecology of the lake has been extensively studied for at least 25 years, a comprehensive picture of the microbial food web linking the bacterial layer to phytoplankton and viruses, with explicit measures of primary and secondary production, is still missing. This study sought to understand better the abundances and productivity of microbes in the context of nutrient biogeochemical cycling across the stratified zones of Lake Cadagno. Photosynthetic pigments and chloroplast 16S rRNA gene phylogenies suggested the presence of eukaryotic phytoplankton through the water column. Evidence supported high abundances of Ankyra judayi, a high-alpine adapted chlorophyte, in the oxic mixolimnion where oxygenic-primary production peaked. Through the low- and no-oxygen chemocline and monimolimnion, chlorophytes related to Closteriopsis acicularis, a known genus of meromictic lakes, and Parachlorella kessleri were observed. Chromatium, anoxygenic phototrophic sulfur bacteria, dominated the chemocline along with Lentimicrobium, a genus of known fermenters whose abundance was newly reported in Lake Cadagno. Secondary production peaked in the chemocline suggesting primary producers depend on heterotrophs for nutrient remineralization. As previously observed, sulfur-reducing bacteria (SRBs), especially Desulfocapsa and Desulfobulbus, were present in the chemocline and anoxic monimolimnion. Virus-to-microbe ratios (VMR) peaked in the zone of phytoplankton yet were at a minimum at the peak of Chromatium. These dynamic trends suggest viruses may play a role in the modulation of oxygenic and anoxygenic photo- and chemosynthesis in Lake Cadagno and other permanently stratified systems.ImportanceAs a window to the past, the study offers insights into the role of microbial guilds of Proterozoic ocean chemoclines in the production and recycling of organic matter of sulfur- and ammonia-containing ancient oceans. The new observations described here suggest that eukaryotic algae were persistent in the low oxygen upper-chemocline in association with purple and green sulfur bacteria in the lower half of the chemocline. Further, this study provides the first insights into Lake Cadagno viral ecology. High viral abundances suggested viruses may be essential components of the chemocline where their activity may result in the release and recycling of organic matter. The framework developed in this study through the integration of diverse geochemical and biological data types lays the foundation for future studies to quantitatively resolve the processes performed by discrete populations comprising the microbial loop in this early anoxic ocean analogue.

Microbial consumption of phytoplankton-derived organic carbon in the pelagic food web is an important component of the global C cycle. We studied C cycling in two phytoplankton-bacteria systems (non-axenic cultures of a dinoflagellate Apocalathium malmogiense and a cryptophyte Rhodomonas marina) in two experiments. In the first experiment we grew phytoplankton and bacteria in nutrient replete conditions and followed C processing at early exponential growth phase and at two later phases. Primary production and total community respiration were up to 4 and 7 times higher, respectively, in the A. malmogiense treatments. Based on the optical signals, accumulating dissolved organic C (DOC) was degraded more in the R. marina treatments and the rate of bacterial production to primary production was higher. Thus, the flow of C from phytoplankton to bacteria was relatively higher in R. marina treatments than in A. malmogiense treatments which was further supported by faster 14C transfer from phytoplankton to bacterial biomass. In the second experiment we investigated consumption of the phytoplankton-derived DOC by bacteria. DOC consumption and transformation, bacterial production and bacterial respiration were all higher in R. marina treatments. In both experiments A. malmogiense supported a bacterial community predominated by bacteria specialized in the utilization of less labile DOC (class Bacteroidia) whereas R. marina supported a community predominated by copiotrophic Alpha- and Gammaproteobacteria. Our findings suggest that large dinoflagellates cycle relatively more C between phytoplankton biomass and the inorganic C pool whereas small cryptophytes direct relatively more C to the microbial loop.

Cyclophosphamide (CP) is a chemotherapy drug which is widely used in the treatment of neoplastic diseases and have often been detected in urban and hospital wastewater, and surface waters. However, at present the effects of CP on aquatic organisms and ecosystems are poorly understood. The main objective of the present study was to assess the effect of CP on the structure and functioning of a sub-tropical freshwater ecosystem (macroinvertebrates, zooplankton and phytoplankton) at environmental relevant concentrations. CP (0, 0.5, 5 and 50 μg/L) was applied weekly to 13,600 L mesocosms over a period of four weeks followed by a one month post exposure period. CP was found to dissipate much faster than previous reported in literature and the half-dissipation times were treatment dependent, being 2.2, 21.3 and 23.6 days in the lowest, middle and highest treatments respectively. Only treatment related effects were observed on the community structure at individual samplings with zooplankton (NOECcommunity = 0.5 μg/L) responding at lower concentrations than phytoplankton (NOECcommunity = 5 μg/L) and macroinvertebrates (NOECcommunity ≥ 50 μg/L). The dissolved organic carbon concentration was consistently higher in the 2 highest treatments, indicating a potential effect on food web interactions and/or the microbial loop. At the population level, consistent adverse effects were observed for the plankton taxa Pleuroxus laevis, Dissotrocha sp. and Oscillatoria sp. at all CP concentrations (NOEC

Size-structured food webs form integrated trophic systems where energy is channeled from small to large consumers. Empirical evidence suggests that size structure prevails in aquatic ecosystems, whereas in terrestrial food webs trophic position is largely independent of body size. Compartmentalization of energy channeling according to size classes of consumers was suggested as a mechanism that underpins functioning and stability of terrestrial food webs including those belowground, but their structure has not been empirically assessed across the whole size spectrum. Here we used stable isotope analysis and metabolic regressions to describe size structure and energy use in eight belowground communities with consumers spanning 12 orders of magnitude in living body mass, from protists to earthworms. We showed a negative correlation between trophic position and body mass in invertebrate communities and a remarkable nonlinearity in community metabolism and trophic positions across all size classes. Specifically, we found that the correlation between body mass and trophic level is positive in the small-sized (protists, nematodes, arthropods below 1 μg in body mass), neutral in the medium-sized (arthropods of 1 μg to 1 mg), and negative in the large-sized consumers (large arthropods, earthworms), suggesting that these groups form compartments with different trophic organization. Based on this pattern, we propose a concept of belowground food webs being composed of (1) size-structured micro-food web driving fast energy channeling and nutrient release, for example in microbial loop; (2) arthropod macro-food web with no clear correlation between body size and trophic level, hosting soil arthropod diversity and subsidizing aboveground predators; and (3) "trophic whales," sequestering energy in their large bodies and restricting its propagation to higher trophic levels in belowground food webs. The three size compartments are based on a similar set of basal resources, but contribute to different ecosystem-level functions and respond differently to variations in climate, soil characteristics and land use. We suggest that the widely used vision of resource-based energy channeling in belowground food webs can be complemented with size-based energy channeling, where ecosystem multifunctionality, biodiversity, and stability are supported by a balance across individual size compartments.

In the context of global warming, changes in phytoplankton-associated bacterial communities have the potential to change biogeochemical cycling and food webs in marine ecosystems. Skeletonema is a cosmopolitan diatom genus in coastal waters worldwide. Here, we grew a Skeletonema strain with its native bacterial assemblage at different temperatures and examined cell concentrations of Skeletonema sp. and free-living bacteria, dissolved organic carbon (DOC) concentrations of cultures, and the community structure of both free-living and attached bacteria at different culture stages. The results showed that elevated temperature increased the specific growth rates of both Skeletonema and free-living bacteria. Different growth stages had a more pronounced effect on community structure compared with temperatures and different physical states of bacteria. The effects of temperature on the structure of the free-living bacterial community were more pronounced compared with diatom-attached bacteria. Carbon metabolism genes and those for some specific amino acid pathways were found to be positively correlated with elevated temperature, which may have profound implications on the oceanic carbon cycle and the marine microbial loop. Network analysis revealed evidence of enhanced cooperation with an increase in positive interactions among different bacteria at elevated temperature. This may help the whole community to overcome the stress of elevated temperature. We speculate that different bacterial species may build more integrated networks with a modified functional profile of the whole community to cope with elevated temperature. This study contributes to an improved understanding of the response of diatom-associated bacterial communities to elevated temperature.

Phytoplankton transform inorganic carbon into thousands of biomolecules that represent an important pool of fixed carbon, nitrogen, and sulfur in the surface ocean. Metabolite production differs between phytoplankton, and the flux of these molecules through the microbial food web depends on compound-specific bioavailability to members of a wider microbial community. Yet relatively little is known about the diversity or concentration of metabolites within marine plankton. Here we compare 313 polar metabolites in 21 cultured phytoplankton species and in natural planktonic communities across environmental gradients to show that bulk community metabolomes reflect chemical composition of the phytoplankton community. We also show that groups of compounds have similar patterns across space and taxonomy suggesting that the concentrations of these compounds in the environment are controlled by similar sources and sinks. We quantify several compounds in the surface ocean that represent substantial understudied pools of labile carbon. For example, the N-containing metabolite homarine was up to 3% of particulate carbon and is produced in high concentrations by culturedSynechococcus, and S-containing gonyol accumulated up to 2.5 nM in surface particles and likely originates from dinoflagellates. Our results show that phytoplankton composition directly shapes the carbon composition of the surface ocean. Our findings suggest that in order to access these pools of bioavailable carbon, the wider microbial community must be adapted to phytoplankton community composition.IMPORTANCEMicroscopic phytoplankton transform 100 million tons of inorganic carbon into thousands of different organic compounds each day. The structure of each chemical is critical to its biological and ecosystem function, yet, the diversity of biomolecules produced by marine microbial communities remained mainly unexplored, especially small polar molecules which are often considered the currency of the microbial loop. Here we explore the abundance and diversity of small biomolecules in planktonic communities across ecological gradients in the North Pacific and within 21 cultured phytoplankton species. Our work demonstrates that phytoplankton diversity is an important determinant of the chemical composition of the highly bioavailable pool of organic carbon in the ocean, and we highlight understudied yet abundant compounds in both the environment and cultured organisms. These findings add to understanding of how the chemical makeup of phytoplankton shapes marine microbial communities where the ability to sense and use biomolecules depends on the chemical structure.

Significance Planktonic microorganisms interact with each other in multifarious ways, ultimately catalyzing the flow of carbon and energy in diverse aquatic environments. However, crucial links associated with eukaryotic microparasites are still overlooked in planktonic networks. We addressed such links by studying cryptic interactions between parasitic fungi, phytoplankton, and bacteria using a model pathosystem. Our results demonstrate that parasitic fungi profoundly modified microbial interactions through several mechanisms (e.g., transferring photosynthetic carbon to infecting fungi, stimulating bacterial colonization on phytoplankton cells, and altering the community composition of bacteria and their acquisition of photosynthetic carbon). Hence, fungal microparasites can substantially shape the microbially mediated carbon flow at the base of aquatic food webs and should be considered as crucial members within plankton communities., Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi, which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed “fungal shunt” can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton–bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles.

All nutrients that plants absorb have to pass a region of intense interactions between roots, microorganisms and animals, termed the rhizosphere. Plants allocate a great portion of their photosynthetically fixed carbon to root-infecting symbionts, such asmycorrhizal fungi; another part is released as exudates fuelling mainly free-living rhizobacteria. Rhizobacteria are strongly top-down regulated by microfaunal grazers, particularly protozoa. Consequently, beneficial effects of protozoa on plant growth have been assigned to nutrients released from consumed bacterial biomass, that is, the 'microbial loop'. In recent years however, the recognition of bacterial communication networks, the common exchange of microbial signals with roots and the fact that these signals are used to enhance the efflux of carbon from roots have revolutionized our view of rhizosphere processes. Most importantly, effects of rhizobacteria on root architecture seem to be driven in large by protozoan grazers. Protozoan effects on plant root systems stand in sharp contrast to effects of mycorrhizal fungi. Because the regulation of root architecture is a key determinant of nutrient- and water-use efficiency in plants, protozoa provide a model system that may considerably advance our understanding of the mechanisms underlying plant growth and community composition.

The availability of phosphorus limits primary production in large regions of the oceans, and marine microbes use a variety of strategies to overcome this limitation. One strategy is the production of alkaline phosphatase (APase), which allows hydrolysis of larger dissolved organic phosphorus (DOP) compounds in the periplasm or at the cell surface for transport of orthophosphate into the cell. Cell lysis, driven by grazing and viral infection, releases phosphorus-containing cell components, along with active enzymes that could persist after lysis. The importance of this continued enzymatic activity for orthophosphate regeneration is unknown. We used three model bacteria – Escherichia coli K-12 MG1655, Synechococcus sp. WH7803, and Prochlorococcus sp. MED4 – to assess the impact of continued APase activity after cell lysis, via lysozyme treatment, on orthophosphate regeneration. Direct release of orthophosphate scaled with cell size and was reduced under phosphate-starved conditions where APase activity continued for days after lysis. All lysate incubations showed post-lysis orthophosphate generation suggesting phosphatases other than APase maintain activity. Rates of DOP hydrolysis and orthophosphate remineralization varied post-lysis among strains. Escherichia coli K-12 MG1655 rates of remineralization were 0.6 and 1.2 amol cell–1hr–1 under deplete and replete conditions; Synechococcus WH7803 lysates ranged from 0.04 up to 0.3 amol cell–1hr–1 during phosphorus deplete and replete conditions, respectively, while in Prochlorococcus MED4 lysates, rates were stable at 0.001 amol cell–1hr–1 in both conditions. The range of rates of hydrolysis and regeneration underscores the taxonomic and biochemical variability in the process of nutrient regeneration and further highlights the complexity of quantitatively resolving the major fluxes within the microbial loop.

Tannic acid is a secondary compound produced by vascular plants and is a major component of mangrove litterfall. Tannic acid is water soluble, leaches out from mangrove litterfall and contributes to DOC and DON pools in adjacent estuaries. About 50% of the litterfall may be degraded and channelized into the marine microbial loop. The influence of tannic acid on bacterioplankton community structure was tested by setting up laboratory-based barrel experiments. Estuarine water from Stn3 of Sundarbans Biological Observatory Time Series (SBOTS) was enriched with tannic acid, and the change in concentration of dissolved nutrients was determined on a daily basis over a span of 15 days. Concentrations of tannic acid, gallic acid and other dissolved nutrients such as nitrate and ortho-phosphate were determined using a UV-Vis spectrophotometer. Tannic acid significantly affected the concentrations of gallic acid and dissolved nitrate in the barrels. Degradation of tannic acid was tracked by a decrease in concentration of tannic acid and generation of gallic acid. The influence of tannic acid on bacterioplankton community structure was analysed on the start (day 0), intermediate (day 3, day 5, day 7 and day 9) and end (day 15) of the experiment. Bacterioplankton community structure was elucidated by sequencing the V3–V4 region of 16S ribosomal RNA on an Illumina MiSeq platform. Proteobacteria was found to be the most dominant bacterial phylum in control and tannic acid–enriched barrels (barrels 1 and 2) on day 0. With the progression of experiment, the abundance of Proteobacteria altered significantly in the control barrel indicating the possible role of this phylum in the breakdown of tannic acid within estuarine mangroves. The abundance of Proteobacteria in the tannic acid–enriched barrels remained high, indicating that members of Proteobacteria may be capable of using tannic acid as a source of carbon and nitrogen. Tannic acid appeared to inhibit most of the other bacterioplankton phyla including Actinobacteria, Acidobacteria and Verrucomicrobia that existed in large abundance in the control barrel on day 15 but were almost absent in the tannic acid–enriched barrels. At class level, Bacteroides was found to be present in highest abundance in the tannic acid–enriched barrels. Tannic acid appeared to strongly influence the abundant bacterioplankton phyla and families as indicated by Pearson’s correlation coefficient and non-metric multidimensional scaling ordination plots. Gallic acid is one of the final products of tannic acid degradation. Breakdown of tannic acid could influence the marine nitrogen and carbon cycling by releasing DON and DOC, respectively, into the adjacent estuaries. Information of breakdown and remineralization of components of litterfall such as tannic acid would also be important for calculation of carbon and nitrogen budgets of coastal ecosystems including in mangroves.

Free-living eukaryotic microbes may reduce animal diseases. We evaluated the dynamics by which micrograzers (primarily protozoa) apply top-down control on the chytrid Batrachochytrium dendrobatidis (Bd) a devastating, panzootic pathogen of amphibians. Although micrograzers consumed zoospores (∼3 μm), the dispersal stage of chytrids, not all species grew monoxenically on zoospores. However, the ubiquitous ciliate Tetrahymena pyriformis, which likely co-occurs with Bd, grew at near its maximum rate (r = 1.7 d–1). A functional response (ingestion vs. prey abundance) for T. pyriformis, measured using spore-surrogates (microspheres) revealed maximum ingestion (Imax) of 1.63 × 103 zoospores d–1, with a half saturation constant (k) of 5.75 × 103 zoospores ml–1. Using these growth and grazing data we developed and assessed a population model that incorporated chytrid-host and micrograzer dynamics. Simulations using our data and realistic parameters obtained from the literature suggested that micrograzers could control Bd and potentially prevent chytridiomycosis (defined as 104 sporangia host–1). However, simulated inferior micrograzers (0.7 × Imax and 1.5 × k) did not prevent chytridiomycosis, although they ultimately reduced pathogen abundance to below levels resulting in disease. These findings indicate how micrograzer responses can be applied when modeling disease dynamics for Bd and other zoosporic fungi.

Sea surface temperature fronts in the Northeastern Arabian Sea (NEAS) facilitate nutrient enhancement in the surface layers, resulting in high primary production. Such high production areas contribute to the accumulation of fresh organic matter rich in carbohydrates, thereby supporting higher heterotrophic metabolism. Transparent exopolysaccharides (TEP) are one of the carbohydrate polymers released as exudates during primary production or decaying process and their breakdown is mediated by a set of ectoenzymes that includes glucosidases and chitinases. Observations were carried out in different aged NEAS fronts to elucidate the influence of frontal age on the abundance of TEP, bacterial nucleic acid content (high nucleic acid content, HNA and low nucleic acid content, LNA), and ectoenzymatic activities. The fronts were classified as younger and older, depending on their first appearance and sampling day. A clear transition from HNA to LNA bacteria, variations in TEP and related ectoenzymatic activities in the fronts was influenced by the frontal age. The younger fronts were dominated by HNA bacteria, α-glucosidase, and chitinase activity. In contrast, an acceleration in β-glucosidase activity, bacterial production, which was related to TEP concentration, was evident in the older fronts. Further, a significant increase in protist numbers in older fronts was related to effective grazing on HNA bacteria and TEP. The active turnover of TEP by bacteria and protists facilitates strong microbial loop in NEAS frontal regions. Elucidating contribution of such changes in the frontal areas will provide a basis for a better understanding of microbial carbon cycling.

Seasonal blooms of the invasive ctenophore Mnemiopsis leidyi have been recorded throughout the Adriatic Sea, affecting the ecosystem and its pelagic resources. Gelatinous organisms such as M. leidyi release significant quantities of excreta as labile and surface active dissolved organic carbon (DOC) that supports the microbial activity. The effects of M. leidyi's presence on heterotrophic prokaryotes (HP), cyanobacteria (CB) and heterotrophic nanoflagellates (HNF) were investigated at five locations in the coastal waters of Rovinj-Rovigno (northern Adriatic) during intensive blooms in September 2019. Additionally, circulation in the study area was examined using available current measurements and a hydrodynamic model. In September, low-pass filtered currents near the surface were mostly directed toward S-SE reaching 25 cm/s, indicating the presence of the Istrian Coastal Countercurrent (ICCC). Ctenophores displayed a patchy spatial distribution from 0.05 to 100 M. leidyi individuals per m3. When ctenophore density in the swarms surpassed 1 individual per m3, the change in the abundances of microbial groups became evident. The lowest microbial densities and lowest concentrations of dissolved and particulate organic carbon (DOC and POC) and surface-active substances occurred in the absence of M. leidyi, while the highest were within the swarms. We hypothesise that HP utilises the available DOC enhancing the remineralisation of organic matter. Finally, greater availability of HNF's prey (HP and CB) leads to an increase in their abundance. This study illustrates how the release of surface-active DOC during intensive M. leidyi's blooms can stimulate the microbial loop. The current system with the ICCC prevailing in September enables the spreading of M. leidyi from its hypothetic origin in an open sea gyre towards the coast. Bora wind episodes may initiate and favour this type of circulation. In these conditions, M. leidyi agglomerates in large numbers along the coast, enabling the accumulation of organic matter and altering the abundance of pico- and nanoplankton.

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.

Nitrogen availability often limits biological productivity in marine systems, where inorganic nitrogen, such as ammonium is assimilated into the food web by bacteria and photoautotrophic eukaryotes. Recently, ammonium assimilation was observed in kleptoplast-containing protists of the phylum foraminifera, possibly via the glutamine synthetase/glutamate synthase (GS/GOGAT) assimilation pathway imported with the kleptoplasts. However, it is not known if the ubiquitous and diverse heterotrophic protists have an innate ability for ammonium assimilation. Using stable isotope incubations (15N-ammonium and 13C-bicarbonate) and combining transmission electron microscopy (TEM) with quantitative nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, we investigated the uptake and assimilation of dissolved inorganic ammonium by two heterotrophic foraminifera; a non-kleptoplastic benthic species, Ammonia sp., and a planktonic species, Globigerina bulloides. These species are heterotrophic and not capable of photosynthesis. Accordingly, they did not assimilate 13C-bicarbonate. However, both species assimilated dissolved 15N-ammonium and incorporated it into organelles of direct importance for ontogenetic growth and development of the cell. These observations demonstrate that at least some heterotrophic protists have an innate cellular mechanism for inorganic ammonium assimilation, highlighting a newly discovered pathway for dissolved inorganic nitrogen (DIN) assimilation within the marine microbial loop.

Organic ligands such as exopolymeric substances (EPS) are known to form complexes with iron (Fe) and modulate phytoplankton growth. However, the effect of organic ligands on bacterial and viral communities remains largely unknown. Here, we assessed how Fe associated with organic ligands influences phytoplankton, microbial, and viral abundances and their diversity in the Southern Ocean. While the particulate organic carbon (POC) was modulated by Fe chemistry and bioavailability in the Drake Passage, the abundance and diversity of microbes and viruses were not governed by Fe bioavailability. Only following amendments with bacterial EPS did bacterial abundances increase, while phenotypic alpha diversity of bacterial and viral communities decreased. The latter was accompanied by significantly enhanced POC, pointing toward the relief of C limitation or other drivers of the microbial loop. Based on the literature and our findings, we propose a conceptual framework by which EPS may affect phytoplankton, bacteria, and viruses. Given the importance of the Southern Ocean for Earth&rsquo, s climate as well as the prevalence of viruses and their increasingly recognized impact on marine biogeochemistry and C cycling, the role of microbe&ndash, virus interactions on primary productivity in the Southern Ocean needs urgent attention.

Particulate organic matter (POM) (fecal pellets) from zooplankton has been demonstrated to be an important nutrient source for the pelagic prokaryotic community. Significantly less is known about the chemical composition of the dissolved organic matter (DOM) produced by these eukaryotes and its influence on pelagic ecosystem structure. Zooplankton migrators, which daily transport surface-derived compounds to depth, may act as important vectors of limiting nutrients for mesopelagic microbial communities. In this role, zooplankton may increase the DOM remineralization rate by heterotrophic prokaryotes through the creation of nutrient rich “hot spots” that could potentially increase niche diversity. To explore these interactions, we collected the migratory copepod Pleuromamma xiphias from the northwestern Sargasso Sea and sampled its excreta after 12–16 h of incubation. We measured bulk dissolved organic carbon (DOC), dissolved free amino acids (DFAA) via high performance liquid chromatography and dissolved targeted metabolites via quantitative mass spectrometry (UPLC-ESI-MSMS) to quantify organic zooplankton excreta production and characterize its composition. We observed production of labile DOM, including amino acids, vitamins, and nucleosides. Additionally, we harvested a portion of the excreta and subsequently used it as the growth medium for mesopelagic (200 m) bacterioplankton dilution cultures. In zooplankton excreta treatments we observed a four-fold increase in bacterioplankton cell densities that reached stationary growth phase after five days of dark incubation. Analyses of 16S rRNA gene amplicons suggested a shift from oligotrophs typical of open ocean and mesopelagic prokaryotic communities to more copiotrophic bacterial lineages in the presence of zooplankton excreta. These results support the hypothesis that zooplankton and prokaryotes are engaged in complex and indirect ecological interactions, broadening our understanding of the microbial loop.

IntroductionThe ability for ecosystems to maintain productivity and functionality under seasonal and long term changes in resource availability relies on the diversity of functional groups (Cadotte et al. 2011). In marine food webs, functionally diverse assemblages of heterotrophic bacteria, heterotrophic protists and zooplankton transfer the organic matter from primary producers to higher trophic levels (Sommer 1989). Zooplankton regulate the flow of energy and matter in the food web through several mechanisms including grazing, respiration, excretion, and as food to support higher trophic levels (Calbet & Landry 2004; Mitra & Davis 2010; Steinberg & Landry 2017). Variation in temporal abundance, feeding traits, size, phenotypic plasticity, growth rate and predation resistance all contribute to the total diversity of zooplankton functional groups in marine food webs (Petchey & Gaston 2006). A high diversity of functional groups contributes to a large variety of resource use that is crucial for the maintenance of ecosystem services under changing conditions (Cadotte et al. 2011). To generate accurate predictions of vulnerability and estimate the resilience of marine ecosystems, a mechanistic understanding of resource use by zooplankton is needed (Bindoff et al. 2019). However, most trophic studies are based on size or phylogeny, and the complexity of zooplankton interactions is rarely considered in trophic studies because of the lack of detailed information about feeding interactions in nature. Consequently, the functional diversity of the zooplankton community and their ability to exploit similar resources is typically not accurately considered (Mitra et al. 2014).The diversity of zooplankton allows for maintaining the biomass of fish stocks over the seasons by a shift from a phytoplankton to detritus-based food webs at times when the biomass of phytoplankton is low or inedible (D’Alelio et al. 2016). While crustacean zooplankton (e.g. copepods and cladocerans) constitute the primary link between phytoplankton and planktivorous fish (Cushing 1990), microzooplankton (i.e. heterotrophic flagellates, ciliates and rotifers) can at times dominate ocean’s carbon respiration in productive coastal ecosystems(Sherr & Sherr 2002; Calbet & Landry 2004). By utilizing matter recycled by heterotrophic bacteria in the microbial loop (Azamet al. 1983), the microzooplankton serve as an additional link between primary producers and crustacean zooplankton (Gifford 1991). The possibility to switch between alternative food web states may be particularly critical in coastal ecosystems that experience an increase in filamentous cyanobacteria due to climate warming (Paerl & Huisman 2008; Cloern et al. 2016).While most trophic studies have clustered zooplankton into broad phylogenetic groups (Mitra et al. 2014), recent studies suggest that models based on traits, particularly size, reflect the true ecosystem structure more effectively (Sommer & Stibor 2002; Boyceet al. 2015). However, none of these approaches consider the entire functional group diversity of zooplankton. As an example, the rotifer phylum contains members of different size classes (belonging to both the micro- and mesozooplankton) (Arndt 1993), as well as organisms with different feeding behaviors such as micro-filtering feeders (Pourriot 1977), selective feeders (Bogdan et al. 1980; Bogdan & Gilbert 1982; Gilbert & Jack 1993), and in some cases even carnivores (Gilbert 1980). Similarly, copepods and cladocerans can perform different feeding strategies ranging from feeding-current feeding to passive/active ambush feeding (Kiorboe 2011), utilizing a wide spectrum of resources.The difficulty in resolving plankton food webs lies within method limitations. Traditional methods to study plankton food webs, such as grazing dilution techniques (Landry & Hassett 1982), biogeochemical tracers or microscopic observations (Post 2002), may not display its full complexity with enough resolution, and has created a biased knowledge towards larger organisms in the food web that are more frequently studied (Gutierrez-Rodriguez et al. 2014). Molecular techniques, including DNA sequencing targeting plankton communities, has highlighted the complexity and diversity of plankton interactions on a global scale (Lima-Mendez et al. 2015). Further, DNA metabarcoding of gut content or selected organisms has proven to be a useful tool for resolving trophic interactions (Pompanon et al.2012) and for zooplankton, barcoding of whole organisms can resolve both trophic, parasitic and mutualistic interactions among crustacean zooplankton (De Corte et al. 2017; Zamora-Terol et al.2020). To our knowledge no study has so far aimed to estimate the diversity of functional groups of zooplankton spanning both phylum and size, using targeted DNA metabarcoding.In this study, we aimed to investigate the functional group diversity of the most abundant zooplankton genera in the Baltic Sea, a temperate coastal sea with strong seasonal variability, and where both micro- and mesozooplankton are at times dominating with well-defined abundance peaks (Fig. 1). We hypothesize that diet composition between zooplankton consumers constitutes a more realistic proxy for functional diversity compared to size and phylogeny. By sequencing 18S rRNA and16S rRNA barcoding genes, we analyzed zooplankton-associated prey of selected individuals of different size classes including a ciliate and rotifers, and compared them with the most abundant crustacean zooplankton (copepods and cladocerans). We demonstrate a larger functional group diversity in resource use within zooplankton in the Baltic Sea than previously acknowledged. The functional group diversity goes beyond both phylogenetic diversity and size and is crucial for the understanding of key ecological processes and maintenance of ecosystem functions.

We used linear inverse ecosystem modeling techniques to assimilate data from extensive Lagrangian field experiments into a mass-balance constrained food web for the Gulf of Mexico open-ocean ecosystem. This region is highly oligotrophic, yet Atlantic bluefin tuna (ABT) travel long distances from feeding grounds in the North Atlantic to spawn there. Our results show extensive nutrient regeneration fueling primary productivity (mostly by cyanobacteria and other picophytoplankton) in the upper euphotic zone. The food web is dominated by themicrobial loop (>70% of net primary productivity is respired by heterotrophic bacteria and protists that feed on them). By contrast, herbivorous food web pathways from phytoplankton to metazoan zooplankton process, ECOLATUN, ECOlogía trófica comparativa de LArvas de aTUN rojo atlántico (Thunnus thynnus) de las áreas de puesta del Medterraneo-NO y el Golfo de México., SI

This article presents the results of research into autochthonous bacteriophages, a previously unknown element in the structure of the microbial loop in the ecosystem of deep oligotrophic Lake Baikal. Transmission electron microscopy was used to study the morphological diversity of phages in the water column, surface microlayer, and benthic biofilms formed at the interface between water and geological rocks (marble, granite, mica, quartz, amphibolite, gabbro, and urtite). This article presents data on the dimensional structure of autochthonous bacteriophages, as well as their quantity, seasonal dynamics, and vertical distribution from the surface to the maximum depth (1200 m) when compared with the content of bacteria. Several morphotypes of Baikal autochthonous bacteriophages affecting Pseudomonas aeruginosa have been isolated and characterized by molecular-genetic methods. The complete genomes of giant phage PaBG from the Myoviridae family and phage MD8 from the Siphoviridae family have been determined.

As the major facilitators of the turnover of organic matter in the marine environment, the ability of heterotrophic bacteria to acquire specific compounds within the diverse range of dissolved organic matter will affect the regeneration of essential nutrients such as iron and carbon. TonB-dependent transporters are a prevalent cellular tool in Gram-negative bacteria that allow a relatively high-molecular-weight fraction of organic matter to be directly accessed. However, these transporters are not well characterized in marine bacteria, limiting our understanding of the flow of specific substrates through the marine microbial loop. Here, we characterize the TonB-dependent transporters responsible for iron and carbon acquisition in a representative marine copiotroph and examine their distribution across the genus Alteromonas. We provide evidence that substrate-specific bioavailability is niche specific, particularly for iron complexes, indicating that transport capacity may serve as a significant control on microbial community dynamics and the resultant cycling of organic matter., Iron is an essential micronutrient for all microbial growth in the marine environment, and in heterotrophic bacteria, iron is tightly linked to carbon metabolism due to its central role as a cofactor in enzymes of the respiratory chain. Here, we present the iron- and carbon-regulated transcriptomes of a representative marine copiotroph, Alteromonas macleodii ATCC 27126, and characterize its cellular transport mechanisms. ATCC 27126 has distinct metabolic responses to iron and carbon limitation and, accordingly, uses distinct sets of TonB-dependent transporters for the acquisition of iron and carbon. These distinct sets of TonB-dependent transporters were of a similar number, indicating that the diversity of carbon and iron substrates available to ATCC 27126 is of a similar scale. For the first time in a marine bacterium, we have also identified six characteristic inner membrane permeases for the transport of siderophores via an ATPase-independent mechanism. An examination of the distribution of specific TonB-dependent transporters in 31 genomes across the genus Alteromonas points to niche specialization in transport capacity, particularly for iron. We conclude that the substrate-specific bioavailability of both iron and carbon in the marine environment will likely be a key control on the processing of organic matter through the microbial loop. IMPORTANCE As the major facilitators of the turnover of organic matter in the marine environment, the ability of heterotrophic bacteria to acquire specific compounds within the diverse range of dissolved organic matter will affect the regeneration of essential nutrients such as iron and carbon. TonB-dependent transporters are a prevalent cellular tool in Gram-negative bacteria that allow a relatively high-molecular-weight fraction of organic matter to be directly accessed. However, these transporters are not well characterized in marine bacteria, limiting our understanding of the flow of specific substrates through the marine microbial loop. Here, we characterize the TonB-dependent transporters responsible for iron and carbon acquisition in a representative marine copiotroph and examine their distribution across the genus Alteromonas. We provide evidence that substrate-specific bioavailability is niche specific, particularly for iron complexes, indicating that transport capacity may serve as a significant control on microbial community dynamics and the resultant cycling of organic matter.

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.

Viruses are ubiquitous in nature and have various ecological functions. Despite their very high abundance (up to 1010 g−1), viruses in soil remain disregarded with just a few, mostly descriptive studies published to date. With this review we focus on the probable functioning of viruses in soil and the consequences for microbial life and turnover, related mechanisms of biogeochemical cycling of carbon (C) and nutrients, and long-term C stabilization. We draw on the limited literature for soil, the richer knowledge from aquatic ecosystems and sediments, and evidence from pure culture studies. Evidence from soil and other ecosystems indicate that the vast majority of soil bacteria are infected by phages at any time. Consequently, we introduce and adapt five concepts for the role of phages in soil: (1) Viral Shunt, (2) ‘Forever Young’, (3) Viral Regulatory Gate of EXOMET, (4) C sequestration by microbial necromass stabilization, and (5) Microscale divergence of C/N/P stoichiometry. The ‘Viral Shunt’ accounts for the short-circuiting of trophic C and nutrient transfers by virus-induced mortality. Phages (even without soil animals – the Microbial Loop concept) explain bacterial death rates and the release of easily available C and nutrients, consequently accelerating biogeochemical cycles in soil. The concept ‘Forever Young’ postulates that viral infection maintains the active bacterial population at a young age, because infection and lysis lead to a short life expectancy. We controversially discuss this concept in relation to hypotheses such as (1) the dormancy of most soil microorganisms, (2) maintenance energy for microorganisms, and (3) their high C use efficiency. We unify the previously suggested but unexplained concepts of ‘Regulatory Gate’ and ‘EXOMET’ through the lytic release of intracellular enzymes and metabolites. Due to the elemental composition of phages, infection results in Stoichiometric C/N/P Imbalances at the microscale, with consequences for P limitation at macroscales. The recently developed concept of ‘C Sequestration by Necromass Stabilization’, i.e., that C sequestered in soil derives from microbial necromass, is discussed from the new perspective of viral lysis and “entombment” of cell fragments in nano-pores. Very few studies have investigated viruses in soil, so all research directions are open, important and fascinating. The most urgent are those elucidating virus functions, their consequences for microbial life, and ecological relevance, and to confirm (or to reject) the proposed concepts. Functioning very fast at the nano-scale, the undead drivers govern microbial life in soil and biogeochemical turnover from micro to ecosystem scales.

The verification that many phytoflagellates ingest prokaryotes has changed the view of the microbial loop in aquatic ecosystems. Still, progress is limited because the phagotrophic activity is difficult to quantify in natural assemblages. Linking the food vacuole content in protist with the ingestion rate of prokaryotes would provide a crucial step forward. In this study, using the catalysed reporter deposition - fluorescence in situ hybridization protocol (CARD-FISH), which allows the visualization of labelled prokaryotes inside protists without relying on incubation procedures, we experimentally relate the food vacuole content of prokaryotes (Vc ) to the population-averaged ingestion rates (Ir ) estimated using bacteria-size fluorescent microspheres. The two variables relate according to the equation Ir = 7.52 Vc 0.9 , which indicates a prokaryote half-life of about 6 min in the protist vacuole. Five mixotrophic flagellate species from natural and culture populations were evaluated seven times during 24 h; they provided a broad range of average vacuole content (0.01 to 2.02 prokaryote protist-1 ) and ingestion rates (0.18 to 23 prokaryote protist-1 h-1 ). Consequently, the relationship found can be applied to quantify the mixotrophy activity in a large variety of field and experimental studies.

Micro glucose biosensors were used to measure net extracellular glucose produced by natural microphytobenthos and three diatom cultures (Amphora coffeaeformis, Navicula menisculus, Nitzschia longissima) from southern Tasmania, Australia. They were exposed to a light gradient in either nutrient‐replete or nutrient‐limiting conditions. Glucose exudation in the natural communities increased with increased light but the response in the cultures was variable. Similarly, nutrient‐replete conditions elicited lower rates of glucose exudation in the natural communities but produced variable species‐specific responses in the cultures. Increased glucose exudation mostly correlated with a reduction in maximum quantum yield (Fv/Fm). The same trend was observed in the natural communities for relative maximum electron transfer rates (rETRmax) but responses in the cultures were again variable and species‐specific. Responses of the three species to increased light and nutrient deficiency were variable, although glucose exudation, Fv/Fm and rETRmax was mostly lower in the nutrient‐limited media. In a second set of experiments species/communities were treated with/without antibiotics. In the dark, glucose concentrations in treatments with antibiotics remained unchanged, while in those with bacteria, it fell rapidly. In the sediment communities, glucose consumption in the dark was ∼25% the rate of exudation at the highest light level. In culture, exudation rates were up to 100% greater than those with active bacteria. Rates of glucose consumption in the dark in the antibiotic–treated samples were negligible and up to 104 times lower than those with active bacteria. These results demonstrate the important role extracellular glucose exudation has on maintaining an active microbial loop.

The distribution of zooplankton has been studied in a waterbody whose entire area is occupied by phytocenoses with the dominance of plants of different ecological groups. It is shown that, in the phytocoenosis of the submerged hydrophyte Elodea canadensis, zooplankton is involved in the classical grazing trophic web. The zooplankton community in this phytocoenosis is mainly made up by algaephagous species. In the habitats occupied by emergent plant Carex riparia, zooplankton is more closely associated with the components of the “microbial loop,” where the most abundant groups are predators and bacterio-detritophagous species.

Mechanisms in controlling the growth of heterotrophic bacteria have seldom been explored in the tropical South China Sea (SCS). This study reports the tempo-spatial distribution patterns and the controlling mechanisms of bacterial biomass (BB), production (BP), and specific growth rate (Bμ) from one summer (Jun 2010; 4 transects) and two winter (January and December 2011; one transect each) cruises along the northern SCS-shelf. In summer, all three bacterial variables showed strong gradients with greater readings at the inner-shelf then decreasing seaward. The positive correlations of bacterial production rate (BP) and bacterial specific growth rate (Bμ), with primary production (PP), chlorophyll-a, and dissolved organic carbon observed in summer indicate a high possibility of bottom-up (substrate supply) control. Positive bacterial temperature response was observed in the inner to mid-shelf area in winter. There, Bμ changed proportionally with temperature up to ca. 22°C. The Q10 (the increase of reaction rate for a temperature rise of 10°C) for Bμ was ~4.0, which was in the range reported by coastal studies. Very high BP/PP ratios (summer average: 89 ± 92%; winter average: 131 ± 88%) indicated bacteria carbon demand relied heavily on allochthonous organic carbon sources such as river input and re-suspension processes, and that the SCS-shelf might be net heterotrophic in these two seasons. In winter, BP/PP ratios changed positively with temperature in areas inside the mid-shelf, suggesting that the coastal zone might become a stronger CO2 source during cold season under a warming climate, if anthropogenic loadings of inorganic nutrients and organic matter remain high in the future.

In recent decades, the phytoplankton community in parts of the Baltic Sea has shifted from diatom dominance to co-occurrence of diatoms and dinoflagellates during the spring bloom. We investigated whether this shift affects bacterial production (BP), abundance and community composition (BCC). Two mesocosm experiments were carried out with water from the SW coast of Finland during the winters of 2012 and 2013. The water was collected before the onset of the spring bloom. Natural seawater was used as a control, and various inocula of diatom and dino flagellate cultures were used as treatments. After the phytoplankton bloom development, BP (thymidine: BPT; leucine: BPL) was significantly higher in the diatom treatments than in the controls and dinoflagellate treatments (BPT and BPL in 2012 and BPL in 2013). In 2013, the BCC was significantly different between the diatom and dinoflagellate treatments and there was a temporal shift in both experiments. Alphaproteobacteria predominated in all treatments at the beginning of the experiments and shifted to flavobacterial (2012) and betaproteobacterial predominance (2013) during the chlorophyll a peak. Towards the end of the experiment, Actinobacteria and Betaproteo bacteria predominated in the diatom treatment in 2012, whereas in 2013 Flavobacteriia (all treatments) predominated together with Gammaproteobacteria and Cytophagia (diatom treatments). The results demonstrated that bacterial physiology and community structure are affected by relatively small changes in the phytoplankton community. Thus, the ongoing changes in the phytoplankton community resulting from co-occurrence of diatoms and dinoflagellates may decrease pelagic remineralization of carbon and reduce organic matter fluxes through the microbial loop.

Appendicularians are planktonic marine tunicates with elaborate filter-feeding houses that can efficiently trap particles as small as 0.2 μm. While marine viruses are seldom considered outside their role in disease transmission, we conducted a controlled laboratory experiment to determine if the appendicularian Oikopleura dioica can trap and ingest the Emiliania huxleyi virus (EhV; 160–180 nm diameter). Removal and retention of EhV during 2.5 h and overnight incubations at 15°C were measured using flow cytometry and quantitative polymerase chain reaction specific for the mcp gene of EhV. The fate of retained EhV was tested by quantifying EhV DNA in three biological compartments: house-trapping, ingestion/digestion, and defecation. Clearance rates for EhV varied from approximately 2 mL ind−1 d−1 to 50 mL ind−1 d−1, with highest rates for 4–5 d-old animals. EhV particles were cleared by O. dioica at rates similar to those reported for larger food particles, with mean clearance rates in the 2.5 h incubations ranging from approximately 2 mL ind−1 d−1 to 50 mL ind−1 d−1. This demonstrates efficient virus removal by O. dioica and a previously overlooked link between the microbial loop and the classical marine food web. EhV DNA was readily detectable above background levels in O. dioica houses, gut contents, and faecal pellets, suggesting that appendicularian houses and faecal pellets may contribute to the dispersal of viruses. Furthermore, clearance of EhV and presumably other viruses by O. dioica may be a significant sink for viruses and thus an important factor in regulating the population dynamics of viruses and their hosts.

International audience; Benthic diatoms are relevant indicators of the ecological status of the littoral zone of lakes. Their use as bio-indicators is based on their morphological identification at species level using microscopy which is time consuming, requires taxonomic expertise, and is consequently expensive. To overcome these limitations, a molecular approach for diatom identification has been tested with success in rivers. DNA metabarcoding enables species identification from a standardized DNA barcode and High-Throughput- Sequencing (HTS), using DNA reference library. The suitability of the morphological and molecular approaches to assess the diatom community structure and the ecological status of the littoral zone of the largest deep lake in France (Lake Bourget) was compared. 66 sites were sampled in August 2015 along the shoreline, all around the lake. The composition of diatom assemblages was similar with both morphological and molecular approaches and diatom assemblages were structured by the same environmental factors. However, the ecological status of Lake Bourget differed significantly among approaches since floristic inventories to species level also differed significantly. The main source of this difference was the incompleteness of the DNA reference library. Nevertheless, in a near future, when this constraint will be solved, the use of DNA metabarcoding for biomonitoring purposes seems promising.

Even though occurrence of fungi in several marine environments has been documented, their inclusion within the marine microbial loop is not fully recognized. A major constraint is whether fungi in coastal waters are truly marine or represent transient microorganisms transported from terrestrial environments. We approached this issue by analyzing ambient fungal composition and hydrolytic activity of culturable fungi along a nearshore-offshore gradient in the upwelling ecosystem off central Chile, a region of high marine productivity strongly influenced by river discharges. We detected different communities of fungi in nearshore and offshore waters, with near estuary strains hydrolyzing proteins and carbohydrates faster than those from offshore sites. We conclude that coastal waters off central Chile comprise distinct fungal communities representative of offshore and nearshore environments, and provide new evidence for fungi processing organic matter in coastal ecotones, opening a fresh perspective for disappearance of organics carried by rivers in the coastal ocean.

High-throughput sequencing (HTS) of soil environmental DNA (eDNA) allows assessing the full diversity of soil micro-eukaryotes. The resulting operational taxonomic units (OTUs) can be assigned to potential taxonomic and functional identities using increasingly complete reference databases. HTS of soil eDNA is revealing a high diversity and abundance of potential eukaryovorous protists, thus challenging the paradigm of the predominantly bacterivorous function of soil phagotrophic protists (i.e. microbial loop). Using Illumina sequencing of soil eDNA and targeting the V9 region of the SSU rRNA gene, we investigated the taxonomic and functional diversities, distribution and co-occurrence patterns of soil micro-eukaryotes in three land-use categories: forests, meadows and croplands located in Switzerland. Each OTU was assigned to a broad functional category (phototrophs, phagotrophs, osmotrophs, or parasites). Total OTU richness was similar in the three land-use categories, but community composition differed significantly between forests and other land-uses. The proportion of fungal sequences (especially Basidiomycota) was highest, and phototroph (i.e. soil microalgae) sequences least abundant in forests. Seven OTUs representing phagotrophic protists, together accounting for >25% of all phagotroph sequences, were significantly correlated to the total number of phototroph sequences, thus suggesting algivory. At least three of these OTUs corresponded to known algal predators. These results suggest that beyond plants, soil microalgae represent a functionally significant but rarely considered input of carbon in soils that should be taken into account when modelling soil nutrient cycling.

We investigated bacterial production (BP) and respiration (BR), as well as the physico-chemical properties of the water column, to elucidate the effect of upwelling on heterotrophic bacterial metabolic activities and growth efficiency (BGE) in July 2012 and May 2013 in the Ulleung Basin (UB), East/Japan Sea. The upwelled conditions were characterized by higher chlorophyll-a (Chl-a) concentrations resulting from the upward shift of the nitracline compared to that of the non-upwelled condition. Analyses of the size fractions of Chl-a and pigment composition revealed that large size phytoplankton (> 20 µm), mainly consisting of diatoms, appeared to be the major phytoplankton component. BP and BR were significantly correlated with Chl-a (P 0.05). These results suggest that bacterial metabolic activities are stimulated by the availability of organic resources enhanced by upwelling in the UB. Further statistical analysis showed that the difference in BP and BGE with variations in upwelling intensity were significant (P = 0.018 for BP, P = 0.035 for BGE), but the difference in BR was not significant (P = 0.321). These results suggest that metabolic energy is partitioned more for BP under a strong upwelling condition, i.e. high nutrient and Chl-a conditions. In contrast, the energy generated via respiration was partitioned more for maintaining metabolism rather than for biomass production under weakly or non-upwelled conditions, i.e. stratified and low Chl-a conditions. Overall, our results suggest that any changes in upwelling intensity would significantly affect the carbon cycle associated with the fate of primary production, and the role of the microbial loop in the UB where changes in the intensity and frequency of upwelling associated with climatic changes are in progress.

Resource availability influences marine microbial community structure, suggesting that population-specific resource partitioning defines discrete niches. Identifying how resources are partitioned among populations, thereby characterizing functional guilds within the communities, remains a challenge for microbial ecologists. We used proteomic stable isotope probing (SIP) and NanoSIMS analysis of phylogenetic microarrays (Chip-SIP) along with 16S rRNA gene amplicon and metagenomic sequencing to characterize the assimilation of six 13C-labeled common metabolic substrates and changes in the microbial community structure within surface water collected from Monterey Bay, CA. Both sequencing approaches indicated distinct substrate-specific community shifts. However, observed changes in relative abundance for individual populations did not correlate well with directly measured substrate assimilation. The complementary SIP techniques identified assimilation of all six substrates by diverse taxa, but also revealed differential assimilation of substrates into protein and ribonucleotide biomass between taxa. Substrate assimilation trends indicated significantly conserved resource partitioning among populations within the Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria classes, suggesting that functional guilds within marine microbial communities are phylogenetically cohesive. However, populations within these classes exhibited heterogeneity in biosynthetic activity, which distinguished high-activity copiotrophs from low-activity oligotrophs. These results indicate distinct growth responses between populations that is not apparent by genome sequencing alone.

We characterized variations in bacterioplankton community composition (BCC) in mesocosms subject to three different treatments. Two groups contained fish (group one: Cyprinus carpio; group two: Hypophthalmichthys molitrix); and group three, the untreated mesocosm, was the control. Samples were taken seven times over a 49-d period, and BCC was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR). Results revealed that introduction of C. carpio and H. molitrix had a remarkable impact on the composition of bacterioplankton communities, and the BCC was significantly different between each treatment. Sequencing of DGGE bands revealed that the bacterioplankton community in the different treatment groups was consistent at a taxonomic level, but differed in its abundance. H. molitrix promoted the richness of Alphaproteobacteria and Actinobacteria, while more bands affiliated to Cyanobacteria were detected in C. carpio mesocosms. The redundancy analysis (RDA) result demonstrated that the BCC was closely related to the bottom-up (total phosphorus, chlorophyll a, phytoplankton biomass) and top-down forces (biomass of copepods and cladocera) in C. carpio and control mesocosms, respectively. We found no evidence for top-down regulation of BCC by zooplankton in H. molitrix mesocosms, while grazing by protozoa (heterotrophic nanoflagellates, ciliates) became the major way to regulate BCC. Total bacterioplankton abundances were significantly higher in C. carpio mesocosms because of high nutrient concentration and suspended solids. Our study provided insights into the relationship between fish and bacterioplankton at species level, leading to a deep understanding of the function of the microbial loop and the aquatic ecosystem.