Comprehensive single-PCR 16S and 18S rRNA community analysis validated with mock communities, and estimation of sequencing bias against 18S.

Universal primers for SSU rRNA genes allow profiling of natural communities by simultaneously amplifying templates from Bacteria, Archaea, and Eukaryota in a single PCR reaction. Despite the potential to show relative abundance for all rRNA genes, universal primers are rarely used, due to various concerns including amplicon length variation and its effect on bioinformatic pipelines. We thus developed 16S and 18S rRNA mock communities and a bioinformatic pipeline to validate this approach. Using these mocks, we show that universal primers (515Y/926R) outperformed eukaryote-specific V4 primers in observed versus expected abundance correlations (slope = 0.88 vs. 0.67-0.79), and mock community members with single mismatches to the primer were strongly underestimated (threefold to eightfold). Using field samples, both primers yielded similar 18S beta-diversity patterns (Mantel test, p < 0.001) but differences in relative proportions of many rarer taxa. To test for length biases, we mixed mock communities (16S + 18S) before PCR and found a twofold underestimation of 18S sequences due to sequencing bias. Correcting for the twofold underestimation, we estimate that, in Southern California field samples (1.2-80 µm), there were averages of 35% 18S, 28% chloroplast 16S, and 37% prokaryote 16S rRNA genes. These data demonstrate the potential for universal primers to generate comprehensive microbiome profiles.

Dynamics and interactions of highly resolved marine plankton via automated high-frequency sampling.

Short timescale observations are valuable for understanding microbial ecological processes. We assessed dynamics in relative abundance and potential activities by sequencing the small sub-unit ribosomal RNA gene (rRNA gene) and rRNA molecules (rRNA) of Bacteria, Archaea, and Eukaryota once to twice daily between March 2014 and May 2014 from the surface ocean off Catalina Island, California. Typically Ostreococcus, Braarudosphaera, Teleaulax, and Synechococcus dominated phytoplankton sequences (including chloroplasts) while SAR11, Sulfitobacter, and Fluviicola dominated non-phytoplankton Bacteria and Archaea. We observed short-lived increases of diatoms, mostly Pseudo-nitzschia and Chaetoceros, with quickly responding Bacteria and Archaea including Flavobacteriaceae (Polaribacter & Formosa), Roseovarius, and Euryarchaeota (MGII), notably the exact amplicon sequence variants we observed responding similarly to another diatom bloom nearby, 3 years prior. We observed correlations representing known interactions among abundant phytoplankton rRNA sequences, demonstrating the biogeochemical and ecological relevance of such interactions: (1) The kleptochloroplastidic ciliate Mesodinium 18S rRNA gene sequences and a single Teleaulax taxon (via 16S rRNA gene sequences) were correlated (Spearman r = 0.83) yet uncorrelated to a Teleaulax 18S rRNA gene OTU, or any other taxon (consistent with a kleptochloroplastidic or karyokleptic relationship) and (2) the photosynthetic prymnesiophyte Braarudosphaera bigelowii and two strains of diazotrophic cyanobacterium UCYN-A were correlated and each taxon was also correlated to other taxa, including B. bigelowii to a verrucomicrobium and a dictyochophyte phytoplankter (all r > 0.8). We also report strong correlations (r > 0.7) between various ciliates, bacteria, and phytoplankton, suggesting interactions via currently unknown mechanisms. These data reiterate the utility of high-frequency time series to show rapid microbial reactions to stimuli, and provide new information about in situ dynamics of previously recognized and hypothesized interactions.

Prokaryotic community successions and interactions in marine biofilms: the key role of Flavobacteriia.

Despite clear advances in characterizing marine biofilms, details on their formation and species succession remain scarce particularly during the early stage of development. We investigated the microbial community composition and succession in coastal marine biofilms on plastic. Samples were collected over 75 days of immersion with strengthened samplings during the early stages of biofilm establishment. Biofilm composition was estimated using Illumina Miseq and microbial community interactions were assessed through microbial association network analysis. In silico analyses showed that primers used in most of previous studies considerably underestimated marine biofilm diversity. Unintentionally ignored so far, we showed that Flavobacteriia might be key actors in the functioning of marine biofilms. Gamma-proteobacteria from the genus Oleibacter strongly dominated microbial communities during the first hours of biofilm formation. These pioneer communities were quickly replaced by alpha-proteobacteria and Flavobacteriia. Bacterial communities exhibited fast temporal structure dynamics with taxa displaying rapid increases and declines. A total of 90% of operational taxonomic units (OTUs) were intermittent or ephemeral reinforcing the conclusion that marine biofilms are highly dynamics. With 2/3 of positive significant connections between bacterial OTUs, microbial biofilm communities appear to be more inclined to develop inter-specific cooperation rather than competition and might thus form sets of functional guilds with mutual metabolic exchanges.

Short-term dynamics and interactions of marine protist communities during the spring-summer transition.

We examined the short-term variability, by daily to weekly sampling, of protist assemblages from March to July in surface water of the San Pedro Ocean Time-series station (eastern North Pacific), by V4 Illumina sequencing of the 18S rRNA gene. The sampling period encompassed a spring bloom followed by progression to summer conditions. Several protistan taxa displayed sharp increases and declines, with whole community Bray-Curtis dissimilarities of adjacent days being 66% in March and 40% in May. High initial abundance of parasitic Cercozoa Cryothecomonas longipes and Protaspis grandis coincided with a precipitous decline of blooming Pseudo-nitzschia diatoms, possibly suggesting their massive infection by these parasites; these cercozoans were hardly detectable afterwards. Canonical correspondence analysis indicated a limited predictability of community variability from environmental factors. This indicates that other factors are relevant in explaining changes in protist community composition at short temporal scales, such as interspecific relationships, stochastic processes, mixing with adjacent water, or advection of patches with different protist communities. Association network analysis revealed that interactions between the many parasitic OTUs and other taxa were overwhelmingly positive and suggest that although sometimes parasites may cause a crash of host populations, they may often follow their hosts and do not regularly cause enough mortality to potentially create negative correlations at the daily to weekly time scales we studied.

Contrasting responses of marine bacterial strains exposed to carboxylated single-walled carbon nanotubes.

The potential toxic effects of carboxylated (COOH) single-walled carbon nanotubes (SWNTs) were investigated on the cell growth and viability of two reference (Silicibacter pomeroyi, Oceanospirillum beijerinckii) and two environmental (Vibrio splendidus, Vibrio gigantis) Gram-negative marine bacterial strains. Bacterial cells were exposed to six concentrations of SWNT-COOH, during different incubation times. Our results revealed different sensitivity levels of marine bacterial strains toward SWNT-COOH exposure. A bactericidal effect of SWNT-COOH has been observed only for Vibrio species, with cell loss viability estimated to 86% for V. gigantis and 98% for V. splendidus exposed to 100 µg mL(-1) of SWNT-COOH during 2h. For both Vibrio strains, dead cells were well individualized and no aggregate formation was observed after SWNT-COOH treatment. The toxic effect of SWNT-COOH on O. beijerinckii cells displayed time dependence, with a longer exposure time reducing their specific growth rate by a factor of 1.2. No significant effect of SWNT-COOH concentration or incubation time had been demonstrated on both growth ability and viability of S. pomeroyi, suggesting a stronger resistance capacity of this strain to carbon nanotubes. The analysis of the relative expression of some functional genes involved in stress responses, using the real-time reverse transcriptase PCR, suggests that the cell membrane damage is not the main toxicity mechanism by which SWNT-COOH interacts with marine bacterial strains. Overall, our results show that SWNT-COOH present a strain dependent toxic effect to marine bacteria and that membrane damage is not the main toxicity mechanism of SWNT in these bacteria.

Temporal dynamics and structure of picocyanobacteria and cyanomyoviruses in two large and deep peri-alpine lakes.

We conducted a 1-year survey of the surface waters of two deep peri-alpine lakes, and investigated the abundances and community structure of picocyanobacteria and co-occurring cyanomyophages. Picocyanobacterial abundances ranged between 4.5 × 10(4) and 1.6 × 10(5) cells mL(-1) in Lake Annecy vs. 2.2 × 10(3) and 1.6 × 10(5) cells mL(-1) in Lake Bourget. Cyanomyoviruses ranged between 2.8 × 10(3) and 3.7 × 10(5) copies of g 20 mL(-1) in Lake Annecy vs. between 9.4 × 10(3) and 9.4 × 10(5) copies of g 20 mL(-1) in Lake Bourget. The structures of picocyanobacteria and cyanomyoviruses differed in the two lakes, and a more pronounced dynamic pattern with greater seasonality was observed in Lake Bourget. At the annual scale, there was no relationship between cyanomyovirus and picocyanobacterial abundances or structures, but we could observe that abundances of the two communities covaried in spring in Lake Bourget. We showed that (i) the changes of picocyanobacteria and cyanomyoviruses were caused by the combined effect of several environmental and biological factors the importance of which differed over time and between the lakes, and (ii) the viral control of the picocyanobacterial community was probably relatively weak at the scale of the investigation.

Spatio-temporal changes in the structure of archaeal communities in two deep freshwater lakes.

In this study, we evaluated the driving forces exerted by a large set of environmental and biological parameters on the spatial and temporal dynamics of archaeal community structure in two neighbouring peri-alpine lakes that differ in terms of trophic status. We analysed monthly data from a 2-year sampling period at two depths corresponding to the epi- and hypolimnetic layers. The archaeal communities seemed to be mainly composed of ammonia-oxidizing archaea belonging to the thaumarchaeotal phylum. The spatio-temporal dynamics of these communities were very similar in the two lakes and were characterized by (1) disparities in archaeal community structure in both time and space and (2) no seasonal reproducibility between years. The archaeal communities were regulated by a complex combination of abiotic factors, including temperature, nutrients, chlorophyll a and dissolved oxygen, and biotic factors such as heterotrophic nanoflagellates and ciliates. However, in most cases, these factors explained < 52% of the variance in archaeal community structure, while we showed in a previous study that these factors explained 70-90% of the temporal variance for bacteria. This suggests that Bacteria and Archaea may be influenced by different factors and could occupy different ecological niches despite similar spatio-temporal dynamics.

Bottom-up versus top-down control of hypo- and epilimnion free-living bacterial community structures in two neighboring freshwater lakes.

Bacterioplankton plays a central role in the microbial functioning of lacustrine ecosystems; however, factors that constrain its structural variation are still poorly understood. Here we evaluated the driving forces exerted by a large set of environmental and biological parameters on the temporal and spatial dynamics of free-living bacterial community structures (BCS) in two neighboring perialpine lakes, Lake Bourget and Lake Annecy, which differ in trophic status. We analyzed monthly data from a 1-year sampling period at two depths situated in the epi- and hypolimnia for each lake. Overall, denaturing gradient gel electrophoresis (DGGE) revealed significant differences in the BCS in the two lakes, characterized by a higher number of bands in the oligotrophic ecosystem (i.e., Lake Annecy). The temporal dynamics of BCS differed greatly between depths and lakes, with temporal scale patterns being much longer in the mesotrophic Lake Bourget. Direct-gradient multivariate ordination analyses showed that a complex array of biogeochemical parameters was the driving force behind BCS shifts in both lakes. Our results indicated that 60 to 80% of the variance was explained only by the bottom-up factors in both lakes, indicating the importance of nutrients and organic matter from autotrophic origin in controlling the BCS. Top-down regulation by flagellates together with ciliates or viruses was found only in the hypolimnion and not in the epilimnion for both lakes and explained less than 18% of the bacterial community changes during the year. Our study suggests that the temporal dynamics of the free-living bacterial community structure in deep perialpine lakes are dependent mainly on bottom-up factors and to a lesser extent on top-down factors, whatever the specific environmental conditions of these lakes.

Effect of grazers and viruses on bacterial community structure and production in two contrasting trophic lakes.

BACKGROUND: Over the last 30 years, extensive studies have revealed the crucial roles played by microbes in aquatic ecosystems. It has been shown that bacteria, viruses and protozoan grazers are dominant in terms of abundance and biomass. The frequent interactions between these microbiological compartments are responsible for strong trophic links from dissolved organic matter to higher trophic levels, via heterotrophic bacteria, which form the basis for the important biogeochemical roles of microbial food webs in aquatic ecosystems. To gain a better understanding of the interactions between bacteria, viruses and flagellates in lacustrine ecosystems, we investigated the effect of protistan bacterivory on bacterial abundance, production and structure [determined by 16S rRNA PCR-DGGE], and viral abundance and activity of two lakes of contrasting trophic status. Four experiments were conducted in the oligotrophic Lake Annecy and the mesotrophic Lake Bourget over two seasons (early spring vs. summer) using a fractionation approach. In situ dark vs. light incubations were performed to consider the effects of the different treatments in the presence and absence of phototrophic activity. RESULTS: The presence of grazers (i.e. <5-µm small eukaryotes) affected viral production positively in all experiments, and the stimulation of viral production (compared to the treatment with no eukaryotic predators) was more variable between lakes than between seasons, with the highest value having been recorded in the mesotrophic lake (+30%). Viral lysis and grazing activities acted additively to sustain high bacterial production in all experiments. Nevertheless, the stimulation of bacterial production was more variable between seasons than between lakes, with the highest values obtained in summer (+33.5% and +37.5% in Lakes Bourget and Annecy, respectively). The presence of both predators (nanoflagellates and viruses) did not seem to have a clear influence upon bacterial community structure according to the four experiments. CONCLUSIONS: Our results highlight the importance of a synergistic effect, i.e. the positive influence of grazers on viral activities in sustaining (directly and indirectly) bacterial production and affecting composition, in both oligotrophic and mesotrophic lakes.

Viral abundance, production, decay rates and life strategies (lysogeny versus lysis) in Lake Bourget (France).

We have investigated the ecology of viruses in Lake Bourget (France) from January to August 2008. Data were analysed for viral and bacterial abundance and production, viral decay, frequency of lysogenic cells, the contribution of bacteriophages to prokaryotic mortality and their potential influence on nutrient dynamics. Analyses and experiments were conducted on samples from the epilimnion (2 m) and the hypolimnion (50 m), taken at the reference site of the lake. The abundance of virus-like particles (VLP) varied from 3.4 × 107to 8.2 × 107 VLP ml⁻¹; with the highest numbers and virus-to-bacterium ratio (VBR = 69) recorded in winter. Viral production varied from 3.2 × 104 VLP ml⁻¹ h⁻¹ (July) to 2 × 106 VLP ml⁻¹ h⁻¹ (February and April), and production was lower in the hypolimnion. Viral decay rate reached 0.12-0.15 day⁻¹, and this parameter varied greatly with sampling date and methodology (i.e. KCN versus filtration). Using transmission electron microscopy (TEM) analysis, viral lysis was responsible for 0% (January) to 71% (February) of bacterial mortality, while viral lysis varied between 0% (April) and 53% (January) per day when using a modified dilution approach. Calculated from viral production and burst size, the virus-induced bacterial mortality varied between 0% (January) and 68% (August). A weak relationship was found between the two first methods (TEM versus dilution approach). Interestingly, flow cytometry analysis performed on the dilution experiment samples revealed that the viral impact was mostly on high DNA content bacterial cells whereas grazing, varying between 8.3% (June) and 75.4% (April), was reflected in both HDNA and LDNA cells equally. The lysogenic fraction varied between 0% (spring/summer) and 62% (winter) of total bacterial abundance, and increased slightly with increasing amounts of mitomycin C added. High percentages of lysogenic cells were recorded when bacterial abundance and activity were the lowest. The calculated release of carbon and phosphorus from viral lysis reached up to 56.5 µgC l⁻¹ day⁻¹ (assuming 20 fgC cell⁻¹) and 1.4 µgP l⁻¹ day⁻¹ (assuming 0.5 fgP cell⁻¹), respectively, which may represent a significant fraction of bacterioplankton nutrient demand. This study provides new evidence of the quantitative and functional importance of the virioplankton in the functioning of microbial food webs in peri-alpine lakes. It also highlights methodologically dependent results.

A 2-year assessment of the main environmental factors driving the free-living bacterial community structure in Lake Bourget (France).

Despite the considerable attention that has been paid to bacterioplankton over recent decades, the dynamic of aquatic bacterial community structure is still poorly understood, and long-term studies are particularly lacking. Moreover, how the environment governs diversity patterns remains a key issue in aquatic microbial ecology. In this study, we used denaturing gradient gel electrophoresis of PCR-amplified partial 16S rRNA gene fragments and multivariable statistical approaches to explore the patterns of change in the free-living bacterial community in the mesotrophic and mono-meromictic Lake Bourget (France). A monthly sampling was conducted over two consecutive years (2007 and 2008) and at two different depths characterizing the epi- and hypolimnion of the lake (2 and 50 m, respectively). Temporal shifts in the bacterial community structure followed different patterns according to depth, and no seasonal reproducibility was recorded from 1 year to the next. Our results showed that the bacterial community structure displayed lower diversity at 2 m (22 bands) compared to 50 m (32 bands) and that bacterial community structure dynamics followed dissimilar trends between the two depths. At 2 m, five shifts in the bacterial community structure occurred, with the temporal scale varying between 2 and 8 months whereas, at 50 m, four shifts in the bacterial community structure took place at 50 m, with the temporal scale fluctuating between 3 and 13 months. More than 60% of the bacterial community structure variance was explained by seven variables at 2 m against eight at 50 m. Nutrients (PO(4)-P, NH(4)-N and NO(3)-N) and temperature were responsible for 49.6% of the variance at 2 m whereas these nutrients, with dissolved oxygen and chlorophyll a accounting for 59.6% of the variance at 50 m. Grazing by ciliates played also a critical role on the bacterial community structure at both depths. Our results suggest that the free-living bacterial community structure in the epi- and hypolimnion of Lake Bourget is mainly driven by combined, but differently weighted, top-down and bottom-up factors at 2 and 50 m.

[The viriosphere: which importance in the functioning and evolution of aquatic ecosystems (part II)?] / La viriosphère : quelle place dans le fonctionnement et l'évolution des écosystèmes aquatiques (partie 2) ?

Besides the lethal effect of viruses on the microbial community (typically the bacteria and the phytoplankton), the viral activity can affect significantly not only the structure of this community but also the cycling of carbon and other nutrients within aquatic ecosystems. Due to their activities, viruses could maintain the prokaryotic community diversity by lysing the most dominant populations then allowing the development of the minor groups. Also, viruses constitute the major driver of horizontal gene transfer between bacterial species or genus and therefore they could be the principal actor in the diversification of microbial communities. The viral lysis which induces the transformation of the biomass into dissolved organic matter pool could stimulate the microbial respiration and reduce the transfer of carbon to higher trophic levels as well as the sedimentation of organic particles towards the bottom. At last, aquatic viruses may generate an important interest for applied issues, typically the phagotherapy for aquaculture, even if an important effort remains to be done in the comprehension of the real efficiency of this therapy and the possible underlying resistance phenomena.

[The viriosphere: which importance in the functioning and evolution of aquatic ecosystems?] / La viriosphère: quelle place dans le fonctionnement et l'évolution des écosystèmes aquatiques (partie 1) ?

The mean abundance of viruses in aquatic ecosystems is about 107 per mL, what leads to the estimation that there are approximately 1030 viruses in these systems on earth. These viruses, which are mainly bacteriophages, i.e. viruses infecting prokaryotes, constitute a very dynamical and diverse biological compartment, although still little is known on their ecology in both marine and limnetic systems. Aquatic viral ecology is indeed a relatively recent topic of aquatic sciences. Each day, 1029 viral infections are likely to occur in the ocean and these infections are probably the main cause of microbial mortality. They may also be responsible for the modification and the diversification of the bacterial community structure. Viral lysis is also proposed as a main driving force in the global biogeochemical cycles in the ocean, with the estimated release of 109 tons of carbon per day, a process largely ignored until recently especially when one refers to the modelisation of the ocean functioning or the coupling between the ocean and the atmosphere. Viruses are thus a main actor of ecosystem functioning with multiple potential roles and this is the reason why they interest more and more ecologists, evolutionists or scientists looking for potential therapeutic agents. In the first part of this review, the focus is made on abundance, dynamics and diversity of aquatic viruses and their role as mortality agents of planktonic populations.