Linking Host Prokaryotic Physiology to Viral Lifestyle Dynamics in a Temperate Freshwater Lake (Lake Pavin,France)

In aquatic ecosystems, fluctuations in environmental conditions and prokaryotic host physiological states can strongly affect the dynamics of viral life strategies. The influence of prokaryote physiology and environmental factors on viral replication cycles (lytic and lysogeny) was investigated from April to September 2011 at three different strata (epi, meta, and hypolimnion) in the mixolimnion of deep volcanic temperate freshwater Lake Pavin (France). Overall, the euphotic region (epi and metalimnion) was more dynamic and showed significant variation in microbial standing stocks, prokaryotic physiological state, and viral life strategies compared to the aphotic hypolimnion which was stable within sampled months. The prokaryotic host physiology as inferred from the nucleic acid content of prokaryotic cells (high or low nucleic acid) was strongly regulated by the chlorophyll concentration. The predominance of the high nucleic acid (HNA) prokaryotes (cells) over low nucleic acid (LNA) prokaryotes (cells) in the spring (HNA/LNA?=?1.2) and vice versa in the summer period (HNA/LNA?=?0.4) suggest that the natural prokaryotic communities underwent major shifts in their physiological states during investigated time period. The increase in the percentage of inducible lysogenic prokaryotes in the summer period was associated with the switch in the dominance of LNA over HNA cells, which coincided with the periods of strong resource (nutrient) limitation. This supports the idea that lysogeny represents a maintenance strategy for viruses in unproductive or harsh nutrient/host conditions. A negative correlation of percentage of lysogenic prokaryotes with HNA cell abundance and chlorophyll suggest that lysogenic cycle is closely related to prokaryotic cells which are stressed or starved due to unavailability of resources for its growth and activity. Our results provide support to previous findings that changes in prokaryote physiology are critical for the promotion and establishment of lysogeny in aquatic ecosystems, which are prone to constant environmental fluctuations.     

Antarctic marine bacterioplankton subpopulations discriminated by their apparent content of nucleic acids differ in their response to ecological factors

Bacterial abundances determined in Drake Passage and Bransfield and Gerlache Straits (Antarctica) in the Austral summer ranged from 0.78 to 9.4×10⁵ cells ml⁻¹, and were positively correlated with standing stocks of Chl a. Two bacterial subpopulations were discriminated based in their different levels of green fluorescence and wide angle light scatter (SSC) per cell after SYTO-13 staining for the first time in Antarctic waters. High nucleic acid (HNA) and low nucleic acid (LNA) subpopulations differed considerably in their response to changes in environmental variables. The apparent content of nucleic acids per cell for the HNA subpopulation (FL1-HNA) showed vertical profiles similar to those of Chl a, including the presence of a maximum at the subsurface chlorophyll maximum. FL1-HNA was positively correlated with Chl a. No similar trends were observed for the LNA fraction. HNA and LNA subpopulations differed in the response of the wide angle light scatter signal to environmental factors as well. SSC-HNA decreased strongly with depth and was positively correlated with Chl a. Again, no similar trends were observed for the LNA subpopulation. The percentage of HNA cells (%HNA) ranged between 35.0 and 76.7% and showed a general tendency to increase with depth. This increase seemed to be larger when the stratification of the water column was higher. Differences in grazing pressure could be responsible of the unexpected vertical distribution of HNA cells. Our results shows that in situ LNA and HNA bacterioplankton subpopulations are under different ecological controls and likely to play different trophodynamic roles in Antarctic waters.     

Phylogenetic characterisation of picoplanktonic populations with high and low nucleic acid content in the North Atlantic Ocean

In flow cytometric analyses of marine prokaryotic picoplankton often two populations with distinct differences in their apparent nucleic acid content are discernable, one with a high and one with a low nucleic acid content (HNA and LNA, respectively). In this study we determined the phylogenetic composition of flow cytometrically sorted HNA and LNA populations, collected at six stations along a transect across three oceanic provinces from Iceland to the Azores. Catalysed reporter deposition fluorescence in situ hybridisation (CARD-FISH) analysis of sorted cells revealed distinct differences in phylogenetic composition between the LNA and HNA populations with only little overlap. At all stations the LNA population was dominated by the alphaproteobacterial clade SAR11 (45–74%). Also, Betaproteobacteria were always present at 2–4%. While the LNA composition was rather stable, the HNA populations were composed of distinct phylogenetic clades in the different oceanic provinces of Arctic and Tropics. For example Cyanobacteria dominated the North Atlantic Gyre HNA population (29–44%) with Prochlorococcus as the major clade (34–44%), but were low in Arctic and Polar waters (1% and 5%, respectively). In contrast, Bacteroidetes accounted for the majority of HNA cells in the Polar and Arctic province (26% and 32%, respectively), but were low in the Gyre region (3–10%). The DNA content of the HNA population was about 3.5 times higher than that of the LNA populations. This reflects differences in the genome sizes of closely related cultured representatives of HNA clades (3–6 Mbp) and LNA clades (1.3–1.5 Mbp).     

Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.     

Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.     

More,smaller bacteria in response to ocean's warming?

Heterotrophic bacteria play a major role in organic matter cycling in the ocean. Although the high abundances and relatively fast growth rates of coastal surface bacterioplankton make them suitable sentinels of global change, past analyses have largely overlooked this functional group. Here, time series analysis of a decade of monthly observations in temperate Atlantic coastal waters revealed strong seasonal patterns in the abundance, size and biomass of the ubiquitous flow-cytometric groups of low (LNA) and high nucleic acid (HNA) content bacteria. Over this relatively short period, we also found that bacterioplankton cells were significantly smaller, a trend that is consistent with the hypothesized temperature-driven decrease in body size. Although decadal cell shrinking was observed for both groups, it was only LNA cells that were strongly coherent, with ecological theories linking temperature, abundance and individual size on both the seasonal and interannual scale. We explain this finding because, relative to their HNA counterparts, marine LNA bacteria are less diverse, dominated by members of the SAR11 clade. Temperature manipulation experiments in 2012 confirmed a direct effect of warming on bacterial size. Concurrent with rising temperatures in spring, significant decadal trends of increasing standing stocks (3% per year) accompanied by decreasing mean cell size (−1% per year) suggest a major shift in community structure, with a larger contribution of LNA bacteria to total biomass. The increasing prevalence of these typically oligotrophic taxa may severely impact marine food webs and carbon fluxes by an overall decrease in the efficiency of the biological pump.     

Characterization of heterotrophic prokaryote subgroups in the Sfax coastal solar salterns by combining flow cytometry cell sorting and phylogenetic analysis

Here, we combined flow cytometry (FCM) and phylogenetic analyses after cell sorting to characterize the dominant groups of the prokaryotic assemblages inhabiting two ponds of increasing salinity: a crystallizer pond (TS) with a salinity of 390 g/L, and the non-crystallizer pond (M1) with a salinity of 200 g/L retrieved from the solar saltern of Sfax in Tunisia. As expected, FCM analysis enabled the resolution of high nucleic acid content (HNA) and low nucleic acid content (LNA) prokaryotes. Next, we performed a taxonomic analysis of the bacterial and archaeal communities comprising the two most populated clusters by phylogenetic analyses of 16S rRNA gene clone library. We show for the first time that the presence of HNA and LNA content cells could also be extended to the archaeal populations. Archaea were detected in all M1 and TS samples, whereas representatives of Bacteria were detected only in LNA for M1 and HNA for TS. Although most of the archaeal sequences remained undetermined, other clones were most frequently affiliated to Haloquadratum and Halorubrum. In contrast, most bacterial clones belonged to the Alphaproteobacteria class (Phyllobacterium genus) in M1 samples and to the Bacteroidetes phylum (Sphingobacteria and Salinibacter genus) in TS samples.     

Low and high nucleic acid content bacteria play discrepant roles in response to various carbon supply modes

Planktonic bacteria can be grouped into ‘high nucleic acid content (HNA) bacteria’ and ‘low nucleic acid content (LNA) bacteria.’ Nutrient input modes vary in environments, causing nutrient availability heterogeneity. We incubated them with equal amounts of total glucose added in a continuous/pulsed mode. The pulse-treated LNA bacteria exhibited twice the cell abundance and four times the viability of the continuous-treated LNA, while HNA did not show an adaptation to pulsed treatment. In structural equation modelling, LNA bacteria had higher path coefficients than HNA, between growth and carbon-saving metabolic pathways, intracellular ATP and the inorganic energy storage polymer, polyphosphate, indicating their low-cost growth, and flexible energy storage and utilisation. After incubation, the pulse-treated LNA bacteria contained more proteins and polysaccharides (0.00064, 0.0012 ng cell⁻¹) than the continuous-treated LNA (0.00014, 0.00014 ng cell⁻¹), conferring endurance and rapid response to pulses. Compared to LNA, HNA keystone taxa had stronger correlations with the primary glucose metabolism step, glycolysis, and occupied leading positions to explain the random forest model. They are essential to introduce glucose into the element cycling of the whole community under both treatments. Our work outlines a systematic bacterial response to carbon input.     

Dynamics of prokaryotic picoplankton community in the central and southern Adriatic Sea (Croatia)

This paper addresses the dynamics of the prokaryotic picoplankton community in the coastal and open sea areas of the central Adriatic and in the coastal area of the southern Adriatic. This involved the study, from January to December 2005, of bacteria (total number of non-pigmented bacteria; high nucleic acid content (HNA) bacteria; low nucleic acid content (LNA) bacteria), cyanobacteria (Synechococcus and Prochlorococcus) and heterotrophic nanoflagellates. During the warmer seasons, in the mainly oligotrophic area under investigation into the Adriatic Sea, bacterial densities and bacterial production have shown an increase in values and domination of the LNA group of the bacterial population. In contrast, in those areas influenced by karstic rivers, the domination of HNA bacteria in total abundance of non-pigmented bacteria and high values of bacterial production was estimated throughout the investigated period. Our results show the importance of both HNA and LNA bacterial groups in the total bacterial activity throughout the investigated area. The biomass of bacteria was mostly predominant in the prokaryotic community, while within the autotrophic community Synechococcus biomass mostly predominated. During the warmer seasons, an increase in autotrophic biomass was observed in relation to non-pigmented biomass. The importance of predation in controlling bacteria by heterotrophic nanoflagellates was pronounced during the warmer period and in the coastal areas.     

Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [³H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.     

Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River

Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river.     

Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.     

A comparative study of the cytometric characteristics of high and low nucleic-acid bacterioplankton cells from different aquatic ecosystems

Flow cytometry has revealed the existence of two distinct fractions of bacterioplankton cells, characterized by high and low nucleic acid contents (HNA and LNA cells). Although these fractions seem ubiquitous in aquatic systems, little is known concerning the variation in the cytometric parameters used to characterize them. We have performed cytometric analyses of samples from a wide range of aquatic systems to determine the magnitude and variability in the cytometric characteristics of HNA/LNA. We show that neither group is associated to a fixed level of fluorescence and of light scatter. Rather, the relative position of HNA and LNA in the fluorescence versus side scatter cytograms varies greatly, both within and among ecosystems. Although the cytometric parameters of both groups tend to covary, there is often uncoupling between the two, particularly in light scatter. Our results show that, although the basic HNA/LNA configuration is present in most samples, its cytometric expression changes greatly in different ecosystems and along productivity gradients. The patterns in cytometric parameters do not support the simple, dichotomous view of HNA and LNA as active and inactive cells, or the notion of two distinct and independent communities, but rather suggest that there may be cells that are intrinsic to each fraction, as well as others that may exchange between fractions.     

Community analysis of high- and low-nucleic acid-containing bacteria in NW Mediterranean coastal waters using 16S rDNA pyrosequencing

The ecological significance of the marine bacterial populations distinguishable by flow cytometry on the basis of the fluorescence (FL) of their nucleic acid (NA) content and proxies of cell size (such as side scatter, SSC) remains largely unknown. Some studies have suggested that cells with high NA (HNA) content and high SSC (HS) represent the active members of the community, while the low NA (LNA) cells are inactive members of the same phylogenetic groups. But group-specific activity measurements and phylogenetic assignment after cell sorting have suggested this is not be the case, particularly in open-ocean communities. To test the extent to which the different NA subgroups are similar, and consequently the extent to which they likely have similar ecological and biogeochemical roles in the environment, we analysed the phylogenetic composition of three populations after cell sorting [high NA-high SC (HNA-HS), high NA-low SC (HNA-LS), low NA (LNA)] by 454 pyrosequencing in two contrasting periods of the year in NW Mediterranean coastal waters (BBMO, Blanes Bay Microbial Observatory) where these three populations have recurrent seasonal patterns. Statistical analyses showed that summer and winter samples were significantly different and, importantly, the sorted populations within a sample were composed of different taxa. The majority of taxa were associated with one NA fraction only, and the degree of overlap (i.e. OTUs present simultaneously in 2 fractions) between HNA and LNA and between summer and winter communities was very small. Rhodobacterales, SAR116 and Bacteroidetes contributed primarily to the HNA fraction, whereas other groups such as SAR11 and SAR86 contributed largely to the LNA fractions. Gammaproteobacteria other than SAR86 showed less preference for one particular NA fraction. An increase in diversity was observed from the LNA to the HNA-HS fraction for both sample dates. Our results suggest that, in Blanes Bay, flow cytometric signatures of natural communities track their phylogenetic composition.     

Ultra- and microplankton assemblages as indicators of trophic status in a Mediterranean lagoon

The seasonal abundance distribution of heterotrophic prokaryotes, pico- and nanophytoplankton, was investigated in connection with environmental variables and microplankton abundance at five stations in Ghar El Melh Lagoon (northeastern Tunisia). Flow cytometry analysis of ultraplankton resolved (i) five heterotrophic prokaryote groups labelled LNA1, LNA2 (low nucleic acid content), HNA1, HNA2 and HNA3 (high nucleic acid content) and (ii) at least 14 ultraphytoplankton groups assigned to picoeukaryotes, picoprokaryotes, nanoeukaryotes, cryptophyte-like cells and some unknown communities. Redundancy analysis (RDA) revealed (i) autumn-summer outbreaks of heterotrophic prokaryotes dominated by HNA groups and (ii) winter-summer proliferation of ultraphytoplankton dominated by nanophytoplankton groups. Generalized additive models (GAM) highlighted the role of (i) water temperature and orthophosphate concentrations in heterotrophic prokaryote distribution and (ii) water temperature and salinity in ultraphytoplankton abundance variation. Based on Spearman's rank correlation, significant negative correlations were established between ultra- and microplankton communities suggesting that, through grazing pressure, microplankton may be behind the drastic decrease in ultraplankton abundances in spring.     

Vertical and seasonal variations of bacterioplankton subgroups with different nucleic Acid contents: possible regulation by phosphorus

We used flow cytometry to examine seasonal variations in basin-scale distributions of bacterioplankton in Lake Biwa, Japan, a large mesotrophic freshwater lake with an oxygenated hypolimnion. The bacterial communities were divided into three subgroups: bacteria with very high nucleic acid contents (VHNA bacteria), bacteria with high nucleic acid contents (HNA bacteria), and bacteria with low nucleic acid contents (LNA bacteria). During the thermal stratification period, the relative abundance of VHNA bacteria (%VHNA) increased with depth, while the reverse trend was evident for LNA bacteria. Seasonally, the %VHNA was strongly positively correlated (r = 0.87; P < 0.001) with the concentration of dissolved inorganic phosphorus, but not with the concentration of chlorophyll a. The growth of VHNA bacteria was significantly enhanced by addition of phosphate or phosphate plus glucose but not by addition of glucose alone. Although the growth of VHNA and HNA bacteria generally exceeded that of LNA bacteria, our data also revealed that LNA bacteria grew faster than and were grazed as fast as VHNA bacteria in late August, when nutrient limitation was presumably severe. Based on these results, we hypothesize that in severely P-limited environments such as Lake Biwa, P limitation exerts more severe constraints on the growth of bacterial groups with higher nucleic acid contents, which allows LNA bacteria to be competitive and become an important component of the microbial loop.     

Vertical and Seasonal Variations of Bacterioplankton Subgroups with Different Nucleic Acid Contents: Possible Regulation by Phosphorus

We used flow cytometry to examine seasonal variations in basin-scale distributions of bacterioplankton in Lake Biwa, Japan, a large mesotrophic freshwater lake with an oxygenated hypolimnion. The bacterial communities were divided into three subgroups: bacteria with very high nucleic acid contents (VHNA bacteria), bacteria with high nucleic acid contents (HNA bacteria), and bacteria with low nucleic acid contents (LNA bacteria). During the thermal stratification period, the relative abundance of VHNA bacteria (%VHNA) increased with depth, while the reverse trend was evident for LNA bacteria. Seasonally, the %VHNA was strongly positively correlated (r = 0.87; P < 0.001) with the concentration of dissolved inorganic phosphorus, but not with the concentration of chlorophyll a. The growth of VHNA bacteria was significantly enhanced by addition of phosphate or phosphate plus glucose but not by addition of glucose alone. Although the growth of VHNA and HNA bacteria generally exceeded that of LNA bacteria, our data also revealed that LNA bacteria grew faster than and were grazed as fast as VHNA bacteria in late August, when nutrient limitation was presumably severe. Based on these results, we hypothesize that in severely P-limited environments such as Lake Biwa, P limitation exerts more severe constraints on the growth of bacterial groups with higher nucleic acid contents, which allows LNA bacteria to be competitive and become an important component of the microbial loop.     

Abundance and production of bacteria, and relationship to phytoplankton production, in a large tropical lake (Lake Tanganyika)

1. Abundance and bacterial production (BP) of heterotrophic bacteria (HBact) were measured in the north and south basins of Lake Tanganyika, East Africa, during seasonal sampling series between 2002 and 2007. The major objective of the study was to assess whether BP can supplement phytoplankton particulate primary production (particulate PP) in the pelagic waters, and whether BP and particulate PP are related in this large lake. HBact were enumerated in the 0–100 m surface layer by epifluorescence microscopy and flow cytometry; BP was quantified using ³H‐thymidine incorporation, usually in three mixolimnion layers (0–40, 40–60 and 60–100 m). 2. Flow cytometry allowed three subpopulations to be distinguished: low nucleic acid content bacteria (LNA), high nucleic acid content bacteria (HNA) and Synechococcus‐like picocyanobacteria (PCya). The proportion of HNA was on average 67% of total bacterial abundance, and tended to increase with depth. HBact abundance was between 1.2 × 10⁵ and 4.8 × 10⁶ cells mL⁻¹, and was maximal in the 0–40 m layer (i.e. roughly, the euphotic layer). Using a single conversion factor of 15 fg C cell⁻¹, estimated from biovolume measurements, average HBact biomass (integrated over a 100‐m water column depth) was 1.89 ± 1.05 g C m⁻². 3. Significant differences in BP appeared between seasons, especially in the south basin. The range of BP integrated over the 0–100 m layer was 93–735 mg C m⁻² day⁻¹, and overlapped with the range of particulate PP (150–1687 mg C m⁻² day⁻¹) measured in the same period of time at the same sites. 4. Depth‐integrated BP was significantly correlated to particulate PP and chlorophyll‐a, and BP in the euphotic layer was on average 25% of PP. 5. These results suggest that HBact contribute substantially to the particulate organic carbon available to consumers in Lake Tanganyika, and that BP may be sustained by phytoplankton‐derived organic carbon in the pelagic waters.     

In vivo screening of modified siRNAs for non-specific antiviral effect in a small fish model: number and localization in the strands are important

Small interfering RNAs (siRNAs) are promising new active compounds in gene medicine but the induction of non-specific immune responses following their delivery continues to be a serious problem. With the purpose of avoiding such effects chemically modified siRNAs are tested in screening assay but often only examining the expression of specific immunologically relevant genes in selected cell populations typically blood cells from treated animals or humans. Assays using a relevant physiological state in biological models as read-out are not common. Here we use a fish model where the innate antiviral effect of siRNAs is functionally monitored as reduced mortality in challenge studies involving an interferon sensitive virus. Modifications with locked nucleic acid (LNA), altritol nucleic acid (ANA) and hexitol nucleic acid (HNA) reduced the antiviral protection in this model indicative of altered immunogenicity. For LNA modified siRNAs, the number and localization of modifications in the single strands was found to be important and a correlation between antiviral protection and the thermal stability of siRNAs was found. The previously published sisiRNA will in some sequences, but not all, increase the antiviral effect of siRNAs. The applied fish model represents a potent tool for conducting fast but statistically and scientifically relevant evaluations of chemically optimized siRNAs with respect to non-specific antiviral effects in vivo.     

Contribution of Archaea to total prokaryotic production in the deep Atlantic Ocean

Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic Archaea (Crenarchaeota and Euryarchaeota) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While Euryarchaeota were found to be more dominant in nearsurface waters, Crenarchaeota were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of Crenarchaeota taking up inorganic carbon increased with depth, while Euryarchaeota taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.