Hydrological connectivity determining metacommunity structure of planktonic heterotrophic flagellates

Dispersal potential of organisms is directly related to the hydrological connectivity among habitats. We investigated if the relative role of the environmental and spatial components in structuring the heterotrophic flagellates depends on the degree of hydrological connectivity. Samplings were performed in aquatic environments of the Paraná River floodplain, which differ in their degree of connectivity: lotic environments, connected lakes and isolated lakes (temporarily connected to the main river only during floods). We expect that communities in isolated lakes would be more subject to dispersal limitation, while in connected lakes and lotic environments the communities would be regulated mainly by environmental variables (species sorting). We sampled in the pelagic region of 23 environments during the low water period in 2014. The results of pRDA revealed that the contributions of environmental and spatial factors differed between the types of environments. The greater contribution of the environmental variables in structuring the flagellates metacommunity, regardless of the type of environment, may be related to the elevated dispersal capacity of microorganisms. The spatial component was also significant in the isolated lakes. Our results support the idea that microorganism communities are mainly structured by environmental factors, although the spatial component seems important when lakes are isolated.     

Ecology of planktonic heterotrophic flagellates. A review.

In aquatic environments heterotrophic flagellates are an important component within the microbial loop and the food web, owing to their involvement in the energy transfer and flux and as an intermediate link between bacteria and primary producers, and greater organisms, such as other protists and metazoan consumers. In the microbial loop heterotrophic flagellates highly contribute to fast biomass and nutrient recycling and to the production in aquatic environments. In fact, these protists consume efficiently viruses, bacteria, cyanobacteria and picophytoplankton, and are grazed mainly by other protists, rotifers and small crustaceans. In this paper the knowledge about these unicellular organisms is reviewed, taking into particular account their ecological relationships and trophic role within the plankton community of marine and freshwater environments.     

Diversity patterns and activity of uncultured marine heterotrophic flagellates unveiled with pyrosequencing

Flagellated heterotrophic microeukaryotes have key roles for the functioning of marine ecosystems as they channel large amounts of organic carbon to the upper trophic levels and control the population sizes of bacteria and archaea. Still, we know very little on the diversity patterns of most groups constituting this evolutionary heterogeneous assemblage. Here, we investigate 11 groups of uncultured flagellates known as MArine STramenopiles (MASTs). MASTs are ecologically very important and branch at the base of stramenopiles. We explored the diversity patterns of MASTs using pyrosequencing (18S rDNA) in coastal European waters. We found that MAST groups range from highly to lowly diversified. Pyrosequencing (hereafter ‘454'') allowed us to approach to the limits of taxonomic diversity for all MAST groups, which varied in one order of magnitude (tens to hundreds) in terms of operational taxonomic units (98% similarity). We did not evidence large differences in activity, as indicated by ratios of DNA:RNA-reads. Most groups were strictly planktonic, although we found some groups that were active in sediments and even in anoxic waters. The proportion of reads per size fraction indicated that most groups were composed of very small cells (∼2–5 μm). In addition, phylogenetically different assemblages appeared to be present in different size fractions, depths and geographic zones. Thus, MAST diversity seems to be highly partitioned in spatial scales. Altogether, our results shed light on these ecologically very important but poorly known groups of uncultured marine flagellates.     

Molecular comparisons of freshwater and marine isolates of the same morphospecies of heterotrophic flagellates

Heterotrophic flagellates are key components of all ecosystems. Understanding the patterns of biodiversity of these organisms is thus particularly important. Here we analyzed the intraspecific diversity of 10 morphospecies of heterotrophic flagellates comprising representatives of the Apusozoa (2 morphospecies) and Kinetoplastea (8 morphospecies), all belonging to the most common flagellates with worldwide distribution. Most morphospecies showed a mixing of lineages isolated from diverse habitats, indicating that some lineages of these morphospecies had been able to colonize different habitats several times. Furthermore, our results revealed remarkable levels of genetic divergence within most of the morphospecies studied, underlining the difficulty of correctly determining species by means of morphology alone. Many cryptic or pseudocryptic species seem to occur. Our results revealed clear divergence between marine and freshwater lineages of the morphospecies Ancyromonas sigmoides, showing that freshwater lineages have not been able to colonize marine environments and marine lineages have not been able to colonize freshwater environments for a long time.     

Dissolved organic matter released by two marine heterotrophic bacterial strains and its bioavailability for natural prokaryotic communities

Marine heterotrophic prokaryotes (HP) play a key role in organic matter processing in the ocean; however, the view of HP as dissolved organic matter (DOM) sources remains underexplored. In this study, we quantified and optically characterized the DOM produced by two single marine bacterial strains. We then tested the availability of these DOM sources to in situ Mediterranean Sea HP communities. Two bacterial strains were used: Photobacterium angustum (a copiotrophic gammaproteobacterium) and Sphingopyxis alaskensis (an oligotrophic alphaproteobacterium). When cultivated on glucose as the sole carbon source, the two strains released from 7% to 23% of initial glucose as bacterial derived DOM (B-DOM), the quality of which (as enrichment in humic or protein-like substances) differed between strains. B-DOM induced significant growth and carbon consumption of natural HP communities, suggesting that it was partly labile. However, B-DOM consistently promoted lower prokaryotic growth efficiencies than in situ DOM. In addition, B-DOM changed HP exoenzymatic activities, enhancing aminopeptidase activity when degrading P. angustum DOM, and alkaline phosphatase activity when using S. alaskensis DOM, and promoted differences in HP diversity and composition. DOM produced by HP affects in situ prokaryotic metabolism and diversity, thus changing the pathways for DOM cycling (e.g. respiration over biomass production) in the ocean.     

Indirectly fluorescently labelled flagellates (IFLF): a tool to estimate the predation on free-living heterotrophic flagellates

A procedure was developed to estimate the direct grazing impacton free-living heterotrophic nanoflagellates (HNF). Culturedflagellates were labelled by feeding on brightly fluorescingbacteria (FLB) and then offered as indirectly fluorescentlylabelled flagellates (IFLF) to potential predators of HNF. Thenumber of FLB in the predators' food vacuoles could be convertedinto IFLF uptake and consumption of HNF. This new techniquewas used to study the HNF-ciliate relationship in the pelagiczone of Lake Constance. Three groups of ciliates were detectedas HNF grazers: small representatives of the genus Strobilidium.a small Haltena-like ciliate (probably Halteria grandinella)and a Codonella sp. Tintinnidium sp. group The ingestion ofHNF by these groups of ciliates ranged between 3 and 15, 3 and39, and 3 and 7 HNF ciliate^–1 h^–1; respectively.The IFLF method allows the direct determination of ingestedflagellate prey in the food vacuoles of their predators. Becauseindigenous living prey organisms were used, tracer discriminationcan be reduced.     

土壤含水量驱动土壤病毒和细菌多度及其与土壤异养呼吸关系的变化

土壤微生物是维持陆地生态系统稳定性和功能的重要组成部分。病毒是地球上数量最多的生物实体,也是若干类型生境中微生物数量的重要调节者。因此,了解病毒与微生物的相互作用,对深入认识包括碳循环在内的生态系统过程具有重要意义。在实验室建立土壤微宇宙实验系统,跟踪调查恒定低含水量、恒定高含水量和波动含水量3种水分处理下土壤病毒和细菌多度的变化,以及土壤异养呼吸速率对土壤病毒-细菌相互作用的响应。相较于低水分处理,高水分处理显著增加了病毒多度（P<0.001）和病毒-细菌多度比（P=0.0026）,波动水分处理显著增加了病毒多度（P<0.001）。在高水分处理的土壤微宇宙中,细菌和病毒多度呈现出随时间动荡的信号,即细菌多度表现出增加-降低-增加的趋势,而病毒多度则表现出增加-降低的趋势,且其变化滞后于细菌。土壤异养呼吸速率与土壤含水量（P<0.001）、细菌多度（P=0.0045）和病毒多度（P<0.001）都具有显著的正相关关系。这些结果说明：病毒导致的下行控制可能是细菌多度的重要影响因子,在水分增加情形下,病毒有可能通过加速细菌的更新速率进而加速土壤呼吸。因此,病毒与细菌的相互作用可能是碳循环的重要决定因素。     

Plasmid transfer to indigenous marine bacterial populations by natural transformation

Abstract Horizontal gene transfer among microbial populations has been assumed to occur in the environment, yet direct observations of this phenomenon are rare or limited to observations where the mechanism(s) could not be explicitly determined. Here we demonstrate the transfer of exogenous plasmid DNA to members of indigenous marine bacterial populations by natural transformation, the first report of this process for any natural microbial community. Ten percent of marine bacterial isolates examined were transformed by plasmid DNA while 14% were transformed by chromosomal DNA. Transformation of mixed marine microbial assemblages was observed in 5 of 14 experiments. In every case, acquisition of the plasmid by members of the indigenous flora was accompanied by modification (probably from genetic rearrangement or methylation) that altered its restriction enzyme digestion pattern. Estimation of transformation rates in estuarine environments based upon the distribution of competency and transformation frequencies in isolates and mixed populations ranged from 5 × 10⁻⁴ to 1.5 transformants/1 day. Extrapolation of these rates to ecosystem scales suggests that natural transformation may be an important mechanism for plasmid transfer among marine bacterial communities.     

Effect of dispersed oil on heterotrophic bacterial communities in cold marine waters

Mesocosm studies were conducted to evaluate the effect of dispersed oil on total and heterotrophic bacterial communities of under-ice seawater from the St. Lawrence Estuary. A regular survey of bacterial changes in the oil-contaminated seawater was performed during a two week period. The bacterial community structure was investigated by carrying out 27 morphological and biochemical tests on 168 isolated strains. The results show a detectable but transient response of the bacterial community to crude oil addition. While total bacterial counts were approximately constant during the experiment, dispersed oil induced an increase in heterotrophic bacterial microflora (from 10⁴ to 10⁵ bacteria ml^-1 after two weeks of contamination). The dispersed oil appeared to have an inhibitory effect on some components of the bacterial community. A decrease of most probable number values was observed just after addition of crude oil in the most polluted tanks and one day later in the less polluted tank. However, except for the most polluted tank, this adverse effect disappeared rapidly. While the dispersed oil induced a total disappearance of some components of the bacterial community in the most polluted tank, the structure of the bacterial community in the less polluted tank appeared relatively unchanged after 14 days of contamination.Offprint requests to: Daniel Delille     

Structure,diversity, and catabolic potentialities of aerobic heterotrophic bacterial populations associated with continuous cultures of natural marine phytoplankton

Variations of structure (probable taxonomic generic groups; ecological profiles), diversity (Shannon index, ¯H), and average catabolic potentialities (strain's average exoenzyme equipment, EAI; average carbonaceous compound utilization, UAI) of bacterial populations during two experimental phytoplankton blooms are described and show a certain overall unity. Oligotrophic conditions are characterized by high diversity levels (¯H from 3.60 to 4) and moderate catabolic potentialities (EAI and UAI close to 40%). During phytoplankton exponential growth phase bacteria show an EAI stability, but there is an increase of UAI with maximal values at the beginning of chlorophyll plateau (52–57%) and higher values of diversity (¯H greater than 4). Phytoplankton mortalities appear to cause an EAI increase and a decrease of both UAI and ¯H (1.50 to 2). Vibrio-like organisms seem to be closely related to this period.In spite of these similar patterns, many differences appear between both experiments from a taxonomic point of view, the autumnal population being more diversified than the spring one.The results obtained show the value of simultaneous analysis of these different aspects and of this ecological methodology allowing spatial or temporal comparisons.     

Cosmopolitan heterotrophic microeukaryotes are active bacterial grazers in experimental oil-polluted systems

We investigated the population dynamics and prevailing 18S rDNA phylotypes of microeukaryotes (≤ 10 μm) in microcosms containing seawater from either an unpolluted oligotrophic site or a chronically oil-polluted mesotrophic site of the Aegean Sea, amended with crude oil (100 p.p.m. final concentration) and crude oil plus emulsifier (10 p.p.m. final concentration). The addition of oil alone did not result in an important increase of bacteria or their predators, while the addition of oil and emulsifiers caused an important increase in bacteria followed by nanoflagellate predator response. We observed an important shift in the microeukaryotic community structure, which was characterized by the dominance of the same heterotrophic nanoflagellates in all oil-polluted treatments. Thus, the resulting 18S rDNA phylotypes were dominated (48.1–82.4%) by Paraphysomonas foraminifera in all treatments containing crude oil and crude oil plus emulsifier. The origin of the seawater, i.e. unpolluted versus chronically oil-polluted, had no effect on the dominant eukaryote, suggesting that the ubiquitous P. foraminifera is an effective opportunist in oil-polluted aquatic systems. The next dominant phylotypes were Monosiga brevicollis (≤ 27.0%) and Pseudobodo tremulans (≤ 23.1%). However, the addition of the emulsifier increased the dominance of P. foraminifera but decreased that of M. brevicollis and P. tremulans . Our study revealed that these dominant oil-tolerant eukaryotes, which are commonly found in the marine environments, are important grazers of bacteria and as such their dynamics should be taken into account in bioremediation practices in situ .     

Phased cell division in natural and laboratory populations of marine planktonic diatoms

Phytoplankton samples were collected every 2 h and examined for percentage of dividing cells (doublets) during different times of the day. Thalassiothrix nitzschioids Grunow and Tropidoneis antarctica Grunow, var. polyplasta Gran, showed diurnal peaks in cell division at 1700. Thalassiosira rotula Meunier showed a nocturnal peak in cell division at 0300, while Chaetoceros vanheurckii Gran consistently exhibited low cell division rates with no apparent peaks. A culture of T. rotula kept under a similar L:D cycle to that present in the field (16:8 L:D) showed a diurnal peak in cell division at 1700. It appears from these data and recent literature that several factors are involved in the regulation of phased cell division in phytoplankton, including temperature, L:D cycle, nutrients, and size selective grazing by zooplankton.     

Taxonomic composition and biomass of heterotrophic flagellates in relation to lake trophy and season

1. Taxonomic composition and abundance of heterotrophic flagellates (HF) were studied in 55 lakes with different trophy in northern Germany using a live-counting technique.
2. Mean abundances and biomasses of HF ranged from 169 cells L⁻¹ and 22 μg L⁻¹ in mesotrophic lakes to 2439 cells mL^–1 and 475 μg L⁻¹ in hypertrophic lakes, respectively. Highest values were generally observed in spring, but mesotrophic lakes showed maximum values in early summer.
3. The taxonomic composition of HF was not significantly influenced by lake trophy and season. The major fraction of HF consisted of chrysomonads and Protista incertae sedis; other important groups were choanoflagellates and bicosoecids. The size distribution of HF changed with lake trophy and season, with a higher proportion of large HF (> 10 μm) in hypertrophic lakes and in spring, respectively.
4. Correlation analyses revealed a strong negative impact of cladocerans on total HF biomass and especially on large HF. Ciliates and large bacteria (> 10 μm) were strongly positively correlated with HF biomass; small bacteria (< 2 μm) showed a weak positive correlation.
5. Analyses at the level of species and genera revealed distinct distribution patterns of some taxa. Paraphysomonas , Aulacomonas and Quadricilia as large-bodied HF showed highest abundance in hypertrophic lakes and in spring. Attached taxa (e.g. Monosiga , Salpingoeca amphoridium ) were highly abundant in late summer and autumn, whereas Spumella and Kathablepharis occurred frequently in most samples.     

Measuring microzooplankton grazing on planktonic marine bacteria by its impact on bacterial production

Grazing on planktonic bacteria by microzooplankton was estimated by separating bacteria from the larger plankton with 1m pore Nuclepore filtration and measuring changes in bacteria in filtered and unfiltered samples over 24 hours. In the absence of grazers, bacteria increased linearly. The regression coefficient of linear increase was used to estimatein situ bacterial production. When grazers were present, the changes in bacteria concentration usually took the form of a linear decline, and grazing was estimated by subtracting the regression coefficient of the unfiltered sample from that of the 1m filtrate. Results from the Essex estuary-coastal system of northern Massachusetts show grazing and production at rates that indicate a daily turnover of the standing crop of bacteria, with highest values in mid-estuarine waters. Experiments on the size distribution of grazing showed that microzooplankton from 1–3m were responsible for most of the observed decrease in bacteria. It was suggested that the basic pattern of linear increase of the bacteria in the absence of grazing reflects density-dependent limitation by substrate present at the outset of the incubation and is indicative of a population that has been maintained around the mid-point of the logistic growth curve by grazing.     

Phylogenetic characterization of the heterotrophic bacterial communities inhabiting a marine recirculating aquaculture system

Aims: The aim of the present work was to characterize the heterotrophic bacterial community of a marine recirculating aquaculture system (RAS). Methods and Results: An experimental RAS was sampled for the rearing water (RW) and inside the biofilter. Samples were analysed for bacterial abundances, community structure and composition by using a combination of culture‐dependent and ‐independent techniques. The most represented species detected among biofilter clones was Pseudomonas stutzeri, while Ruegeria spp. and Roseobacter spp. were more abundant among isolates. In comparison, the genera Roseobacter and Ruegeria were well represented in both the biofilter and the RW samples. A variety of possible bacterial pathogens (e.g. Vibrio spp., Erwinia spp. and Coxiella spp.) were also identified in this study. Conclusions: Results revealed that the bacterial community in the RW was quite different to that associated with the biofilter. Moreover, data obtained suggest that the whole bacterial community can be involved in maintaining an effective and a stable rearing environment (shelter effect). Significance and Impact of the Study: Improving the reliability and the sustainability of RAS depends on the correct management of the bacterial populations inside it. This study furnishes more accurate information on the bacterial populations and better clarifies the existing relationships between the bacterial flora in the RW and that associated with the biofilter.     

New method for estimating bacterial cell abundances in natural samples by use of sublimation

We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples, including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert, were heated to a temperature of 500 degrees C for several seconds under reduced pressure. The sublimate was collected on a cold finger, and the amount of adenine released from the samples was then determined by high-performance liquid chromatography with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from approximately 10(5) to 10(9) E. coli cell equivalents per gram. For most of these samples, the sublimation-based cell counts were in agreement with total bacterial counts obtained by traditional DAPI (4,6-diamidino-2-phenylindole) staining.     

Nitrogen fixation by a marine non‐heterocystous cyanobacterium requires a heterotrophic bacterial consort

Cultures of the non‐heterocystous cyanobacterium, Leptolyngbya nodulosa, could be grown indefinitely in media devoid of combined nitrogen. Acetylene reduction assays showed that these cultures fixed nitrogen in the dark period of a diurnal cycle under micro‐oxygenic or anaerobic conditions. Addition of DCMU to cultures induced much higher rates of nitrogenase activity, most of which occurred in the light. Measurements of activity in the presence of chloramphenicol indicated that nitrogenase is synthesized in darkness and probably destroyed in the subsequent light period. Neither the dark‐mediated nitrogenase in the absence of DCMU nor light‐mediated activity in the presence of DCMU could be sustained for more than 3 days without a photoperiodic light/dark cycle. Axenic cultures could not be grown in the absence of combined nitrogen and did not demonstrate any acetylene reduction activity. An identical nifH gene sequence was found in axenic and non‐axenic cultures of L. nodulosa. RT‐PCR demonstrated that this gene was expressed only in non‐axenic cultures. Western blotting showed that the Fe‐protein of nitrogenase is absent in cultures that are incapable of acetylene reduction, indicating that the lack of nitrogenase activity is likely due to the absence of the enzyme. These observations strongly indicate that L. nodulosa contains a functional nitrogenase which is not expressed in the absence of heterotrophic bacteria.     

Effects of bottom-up and top-down controls on the temporal distribution of planktonic heterotrophic nanoflagellates are dependent on water depth

The ecological function of the Mesopotamian marshlands was severely damaged during their desiccation from 1993 to 2003 and, as a result, the marshes became a main target for restoration after their re-flooding in April 2003. In this study, the phosphorus (P) and nitrogen (N) budgets for a portion of the Mesopotamian marshlands at Al-Hawizeh (Iraq) are reported. Al-Hawizeh appears to play a strong role in regulating P and N. Total P input and output were both dominated by dissolved organic P, while total N input and output were dominated by particulate organic N, suggesting that N is in greater demand than P. Al-Hawizeh acts as source for N and sink for P; this has an important function in altering their export downstream (Shatt Al-Arab River) and subsequently into the Arabian Gulf. However, investigating P and N fractions helped in understanding the role of Al-Hawizeh in transforming, storage and release of P and N fractions.