Seasonal Depth-Related Gradients in Virioplankton: Standing Stock and Relationships with Microbial Communities in Lake Pavin (France)

This study presents a depth-related survey of virioplankton abundance in Lake Pavin (Massif Central, France), in relation to the abundances of heterotrophic prokaryotes, picocyanobacteria (Pcy), autotrophic picoeukaryotes (Peu), and of autotrophic (ANF) and heterotrophic (HNF) nanoflagellates. The sampling strategy was designed to be representative of the physico-chemical gradients of the whole water column of the lake, and the seasonal variability as well. In mixolimnic surface waters, all communities were present and viral abundance peaked in summer and autumn. Viral abundance was significantly correlated (p < 0.001) with Pcy, Peu, and ANF, indicating that cyanophages and perhaps other phytoplankton viruses represent a significant pool of viral standing stocks in the mixolimnion of Lake Pavin. Microautotrophs were absent in the deep monimolimnic water masses, where viruses and heterotrophic prokaryotes exhibited highest seasonal abundances in summer and/or autumn and were significantly correlated (p < 0.001) to each other. This indicates that the anoxic monimolimnion of Lake Pavin is an exclusive habitat for viruses and heterotrophic prokaryotes. We conclude that in this habitat, host availability is prevalent over other factors (temperature, oxygen, nutrients, grazers) in favoring viral proliferation.     

Depth-Related Gradients of Viral Activity in Lake Pavin

High-resolution vertical sampling and determination of viral and prokaryotic parameters in a deep volcanic lake shows that in the absence of thermal stratification but within light, oxygen, and chlorophyll gradients, host availability empirically is prevalent over the physical and chemical environments and favors lytic over lysogenic “viral life cycles.”     

Seasonal changes in alkaline phosphatase activity of bacteria and microalgae in Lake Pavin (Massif Central,France)

Seasonal changes in alkalinephosphatase activity of bacteria and microalgae werestudied in the dimictic Lake Pavin (Massif Central,France), to test whetherthis activity is primarily algal or bacterial andwhether the APA presents seasonal variations coupledwith abiotic and biotic variables. Samples werecollected at different depths from May 1992 to May1993. The specific phosphatase activities wereanalysed spectrophotometrically with p-NitrophenylPhosphate (p-NPP) as substrate and were related to theprotein concentrations. No correlation was foundbetween alkaline phosphatase activity and solublereactive phosphorus (SRP) concentrations across anannual cycle. The specific activities of the smallclass (0.2–1.2 m) were the highest and thecontribution of this picoplanktonic size class(0.2–1.2 m) increased with depth. In addition, thelinear correlations between alkaline phosphataseactivity and protein concentration seemed to indicatethat most of these enzymes are constitutive. However,it cannot be excluded that the high phosphorusconcentrations repress APA.Finally, the measure of APA does not seem to be avalid quantitative test of the deficiency ofphosphorus for aquatic microorganisms.     

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.     

Phylogenetic Diversity of Archaea and Bacteria in the Anoxic Zone of a Meromictic Lake (Lake Pavin, France)

The compositions of archaeal and bacterial populations at different depths (60 m [mixolimnion-chemocline interface], 70 m [chemocline-subchemocline interface], 90 m, and 92 m [the water-sediment interface]) in the anoxic zone of the water column in Lake Pavin, a freshwater permanently stratified mountain lake in France, were determined. Phylogenetic trees were constructed from sequences to assess archaeal and bacterial diversity at the four sites.     

Phylogenetic diversity of archaea and bacteria in the anoxic zone of a meromictic lake (Lake Pavin, France)

The compositions of archaeal and bacterial populations at different depths (60 m [mixolimnion-chemocline interface], 70 m [chemocline-subchemocline interface], 90 m, and 92 m [the water-sediment interface]) in the anoxic zone of the water column in Lake Pavin, a freshwater permanently stratified mountain lake in France, were determined. Phylogenetic trees were constructed from sequences to assess archaeal and bacterial diversity at the four sites.     

Dynamic of Virioplankton Abundance and Its Environmental Control in the Charente Estuary (France)

The Charente River provides nutrient- and virus-rich freshwater input to the Marennes Oléron Basin, the largest oyster-producing region in Europe. To evaluate virioplankton distribution in the Charente Estuary and identify which environmental variables control dynamic of virioplankton abundance, five stations defined by a salinity gradient (0–0.5, 0.6–5, 13–17, 20–24, and higher than 30 PSU) were surveyed over a year. Viral abundance was related to bacterioplankton abundance and activities, photosynthetic pigments, nutrient concentration, and physical parameters (temperature and salinity). On a spatial scale, virus displayed a decreasing pattern seaward with abundance ranging over the sampling period from 1.4 × 10⁷ to 20.8 × 10⁷ viruses mL⁻¹ making virioplankton the most abundant component of planktonic microorganisms in the Charente Estuary. A good correlation was found between viral and bacterial abundance (r_s = 0.85). Furthermore, bacterial abundance was the most important predictor of viral abundance explaining alone between 66% (winter) and 76% (summer) of viral variability. However, no relation existed between viral abundance and chlorophyll a. Temporal variations in viral distributions were mainly controlled by temperature through the control of bacterial dynamics. Spatial variations of viral abundance were influenced by hydrodynamic conditions especially during the winter season where virioplankton distribution was entirely driven by mixing processes.     

Reproduction,condition and food of adult arctic charr (Salvelinus alpinus,L.) in Lake Pavin (Massif Central,France)

RésuméTo determine the role and the importance of adult fish predation directly and indirectly in the food web of oligomesotrophic Lake Pavin, reproduction, condition, and feeding activity of the dominant adult chctic charr (Salvelinus alpinus, L.) were studied. Fish were caught by gillnets of different mesh sizes once a month from April to December 1992. The gonad cycle showed that the spawning period occurred between October to December. Relative fecundity is about 2509 ovules per kg of female fish and ovule mean diameter is 4.7 mm ( 0.6 mm). Seasonal variations of the sex-ratio (Max. in April = 2.67 and Min. in October=0.19) indicated the intralake migrations of females and males. The condition coefficients showed higher values for females than males; the maximum was observed in summer for both sexes and the minimum in April and in October for males and females, respectively. Low values of the condition were also observed during the spawning period. High feeding activity was recorded in spring and in December after reproduction; low feeding activity was observed in October during reproduction. Asellus aquaticus was quantitatively the dominant prey from April to September; during the sp awning period, S. alpinus consumed its own eggs, particularly in large quantity in December. Daphnia longispina was numerically the dominant prey all over the studied cycle and the electivity (Ivlev Index) of arctic charr on the latter prey ranged from +s+0.79 to +0.16. During April, more than 15 500 individuals of D. longispina per kg of fish were found in the gut contents. The selective predation of S. alpinus on benthic (A. aquatics) and zooplankton prey (D. longispina) indicated that the fish immigrate during their feeding activity from benthic/littoral to the pelagic area.Afin de d terminer le rôle et l'importance de la prédation directe et indirecte de l'omble chevalier adulte (Salvelinus alpinus, L.) dans le réseau trophique du lac Pavin, milieu oligo- mésotrophe, nous avons étudié la reproduction, deux facteurs de condition et l'activité alimentaire de ce poisson qui est l'espéce dominante dans ce lac. Les poissons ont été capturés par des filets maillants de différent vide de maille selon un pas d'échantillonnage mensuel d'avril á décembre 1992. Le cycle du développement des gonades montre que la période de reproduction se déroule entre octobre et décembre. La fécondité relative calculée est de 2509 ovules par kg de femelles et le diamétre moyen d'un ovule est de 4,7 mm ( 0,6). Les variations saisonniéres du sex-ratio (Max. en avril = 2,67 et Min. en octobre=0,19) indiquent le déplacement actif et successif des femelles et des mâles. Les variations saisonniéres des coefficients de condition indiquent une copulence plus importante des femelles par rapport aux mâles avec des valeurs maximales en été pour les deux sexes et des valeurs minimales en avril et en octobre pour les mâles et les femelles respectivement. De faibles valeurs des facteurs de condition ont été observées pendant la période de reproduction pour les deux sexes. L'activité alimentaire de l'omble chevalier est intense au printemps et au mois de décembre aprés la fraie et faible au mois d'octobre pendant la reproduction. Asellus aquaticus est, en termes de biomasse, la proie dominante d'avril á septembre. Pendant la période de reproduction, S. alpinus se nourrit de ses propres oeufs sur les zones de frayéres et en décembre la quantité d'oeufs consommée par le Salmonidé est importante. Daphnia longispina est, en termes d'abondance, la proie dominante tout au long de cette étude et l'indice d'électivité (Indice d'Ivlev) de l' ombre chevalier vis-á-vis de cette proie varie entre + 0,79 et + 0,16.De plus, au mois d'avril, plus de 15 500 D. longispina par kg de poisson ont été enregistrées dans les contenus stomacaux. La prédation sélective de S. alpinus sur des proies benthiques (A. aquaticus) et zooplanctoniques (D. longispina) indique que le poisson effectue des déplacements en fonction de son activité alimentaire de la zone littorale/benthique á la zone pélagique.     

Photolithotrophy,photoheterotrophy and chemoheterotrophy during spring phytoplankton development (Lake Pavin)

In order to determine the relative importance of autotrophic and heterotrophic activities in both bacterial and phytoplanktonic communities in an oligomesotrophic lake, the size fractionation by differential filtration and the use of a bacterial inhibitor (gentamycin) were combined. The study was carried out at Lake Pavin during the spring planktonic bloom. Photosynthetic and photo- and chemoheterotrophic activities were measured from the assimilation of NaH¹⁴CO₃ and glucose-³H, using a double labeling technique. The bacterial community was at low cell concentration (0.6 to 7 × 10⁵ cells ml^–) and represented very low biomass values (0.9 to 11.5 gC liter^–1). The abundance of the phytoplankton varied between 0.5 and 1.8 × 10⁶ cells liter^–1, and biomass values ranged between 19 and 118 gC liter^–1. The diatom Melosira italica sp. subarctica (O. Mueller) was the largely dominant species in the meta- and hypolimnion. Inorganic fixation by photolithotrophy (mean value: 1.66 mg C m^–3 hour^–1) largely predominates over assimilation by photoheterotrophy (mean value: 0.93 g C m^–3 hour^–1) or chemoheterotrophy (mean value: 2.42 g C m^–3 hour^–1). However, because of the considerable underestimation of heterotrophic assimilation due to the experimental methods used, and because of the spatial and temporal separation of photolithotrophic and photo- and chemoheterotrophic activities, it is likely that the fixation of organic carbon by microalgae plays an important role in the survival of species and/or in competitive interactions, as the results with Monoraphidium contortum (Pasch. et Korschik.), the prevailing species in the epilimnion, would suggest. Send offprint requests to: C. Amblard.     

Protistan diversity in a permanently stratified meromictic lake (Lake Alatsee,SW Germany)

Protists play a crucial role for ecosystem function(ing) and oxygen is one of the strongest barriers against their local dispersal. However, protistan diversity in freshwater habitats with oxygen gradients received very little attention. We applied high‐throughput sequencing of the V9 region (18S rRNA gene) to provide a hitherto unique spatiotemporal analysis of protistan diversity along the oxygen gradient of a freshwater meromictic lake (Lake Alatsee, SW Germany). In the mixolimnion, the communities experienced most seasonal structural changes, with Stramenopiles dominating in autumn and Dinoflagellata in summer. The suboxic interface supported the highest diversity, but only 23 OTUs_95% (mainly Euglenozoa, after quality check and removal of operational taxonomic units (OTUs) with less than three sequences) were exclusively associated with this habitat. Eukaryotic communities in the anoxic monimolimnion showed the most stable seasonal pattern, with Chrysophyta and Bicosoecida being the dominant taxa. Our data pinpoint to the ecological role of the interface as a short‐term ‘meeting point’ for protists, contributing to the coupling of the mixolimnion and the monimolimnion. Our analyses of divergent genetic diversity suggest a high degree of previously undescribed OTUs. Future research will have to reveal if this result actually points to a high number of undescribed species in such freshwater habitats.     

Succession and Regulation Factors of Small Eukaryote Community Composition in a Lacustrine Ecosystem (Lake Pavin)

The structure and dynamics of small eukaryotes (cells with a diameter less than 5 μm) were studied over two consecutive years in an oligomesotrophic lake (Lake Pavin in France). Water samples were collected at 5 and 30 m below the surface; when the lake was stratified, these depths corresponded to the epilimnion and hypolimnion. Changes in small-eukaryote structure were analyzed using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the 18S rRNA genes. Terminal restriction fragments from clones were used to reveal the dominant taxa in T-RFLP profiles of the environmental samples. Spumella-like cells (Chrysophyceae) did not dominate the small eukaryote community identified by molecular techniques in lacustrine ecosystems. Small eukaryotes appeared to be dominated by heterotrophic cells, particularly Cercozoa, which represented nearly half of the identified phylotypes, followed by the Fungi-LKM11 group (25%), choanoflagellates (10.3%) and Chrysophyceae (8.9%). Bicosoecida, Cryptophyta, and ciliates represented less than 9% of the community studied. No seasonal reproducibility in temporal evolution of the small-eukaryote community was observed from 1 year to the next. The T-RFLP patterns were related to bottom-up (resources) and top-down (grazing) variables using canonical correspondence analysis. The results showed a strong top-down regulation of small eukaryotes by zooplankton, more exactly, by cladocerans at 5 m and copepods at 30 m. Among bottom-up factors, temperature had a significant effect at both depths. The concentrations of nitrogenous nutrients and total phosphorus also had an effect on small-eukaryote dynamics at 5 m, whereas bacterial abundance and dissolved oxygen played a more important structuring role in the deeper zone.     

Succession and regulation factors of small eukaryote community composition in a lacustrine ecosystem (Lake Pavin)

The structure and dynamics of small eukaryotes (cells with a diameter less than 5 microm) were studied over two consecutive years in an oligomesotrophic lake (Lake Pavin in France). Water samples were collected at 5 and 30 m below the surface; when the lake was stratified, these depths corresponded to the epilimnion and hypolimnion. Changes in small-eukaryote structure were analyzed using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the 18S rRNA genes. Terminal restriction fragments from clones were used to reveal the dominant taxa in T-RFLP profiles of the environmental samples. Spumella-like cells (Chrysophyceae) did not dominate the small eukaryote community identified by molecular techniques in lacustrine ecosystems. Small eukaryotes appeared to be dominated by heterotrophic cells, particularly Cercozoa, which represented nearly half of the identified phylotypes, followed by the Fungi-LKM11 group (25%), choanoflagellates (10.3%) and Chrysophyceae (8.9%). Bicosoecida, Cryptophyta, and ciliates represented less than 9% of the community studied. No seasonal reproducibility in temporal evolution of the small-eukaryote community was observed from 1 year to the next. The T-RFLP patterns were related to bottom-up (resources) and top-down (grazing) variables using canonical correspondence analysis. The results showed a strong top-down regulation of small eukaryotes by zooplankton, more exactly, by cladocerans at 5 m and copepods at 30 m. Among bottom-up factors, temperature had a significant effect at both depths. The concentrations of nitrogenous nutrients and total phosphorus also had an effect on small-eukaryote dynamics at 5 m, whereas bacterial abundance and dissolved oxygen played a more important structuring role in the deeper zone.     

Members of candidate divisions OP11, OD1 and SR1 are widespread along the water column of the meromictic Lake Pavin (France)

The vertical distribution of OP11, OD1 and SR1 divisions in the oxycline and in the anoxic water column of Lake Pavin, a freshwater permanently stratified mountain lake in France, was determined by temporal temperature gel gradient electrophoresis and 16S rRNA clone libraries. Gradual changes in the community structure were noted in relation to environmental variables along the oxidized/reduced environment. In addition, a separate effort to identify members of these lineages in the oxic mixolimnion identified sequences affiliated to SR1 and OP11 divisions, indicating that they are more widespread than previously expected.     

The physical structure and dynamics of a deep,meromictic crater lake (Lac Pavin,France)

The characteristic feature of the physical structure of Lac Pavin is a distinct and permanent chemically induced density increase between about 60 and 70 m depth. This chemocline separates the seasonally mixed mixolimnion from the monimolimnion, which is characterized by elevated temperature and salinity as well as complete anoxia. Previously published box-models of the lake postulated substantial groundwater input at the lake bottom, and consequently a short water residence time in the monimolimnion and high fluxes of dissolved constituents across the chemocline. We present a new view of the physical structure and dynamics of Lac Pavin, which is based on the results of high-resolution CTD profiles, transient and geochemical tracers (tritium, CFCs, helium), and numerical modeling. The CTD profiles indicate the existence of a sublacustrine spring above rather than below the chemocline. A stability analysis of the water column suggests that vertical turbulent mixing in the chemocline is very weak. A numerical one-dimensional lake model is used to reconstruct the evolution of transient tracer distributions over the past 50 years. Model parameters such as vertical diffusivity and size of sublacustrine springs are constrained by comparison with observed tracer data. Whereas the presence of a significant water input to the monimolimnion can clearly be excluded, the input to the mixolimnion – both at the surface and from the indicated sublacustrine spring – cannot be accurately determined. The vertical turbulent diffusivity in the chemocline is well constrained to K 5×10^-8 m² s^-1, about a factor of three below the molecular diffusivity for heat. Assuming thus purely molecular heat transport, the heat flow through the chemocline can be estimated to between 30 and 40 mW m^-2. With respect to dissolved constituents, the very weak turbulent diffusive exchange is equivalent to a stagnant monimolimnion with a residence time of nearly 100 years. Based on these results and vertical concentration profiles of dissolved species, diffusive fluxes between monimolimnion and mixolimnion can be calculated. A large excess of helium with a ³He/⁴He ratio of (9.09 ± 0.01)×10^-6 (6.57 R _a) is present in the monimolimnion, clearly indicating a flux of magmatic gases into the monimolimnion. We calculate a flux of 1.0×10^-12 mol m^-2 s^-1 for mantle helium and infer a flux of 1.2×10^-7 mol m^-2 s^-1 (72 t year^-1) for magmatic CO₂. The monimolimnion appears to be in steady state with respect to these fluxes.     

Protistan grazing and viral lysis losses of bacterial carbon production in a large mesotrophic lake (Lake Biwa)

The grazing and lysis mortalities of planktonic bacteria were estimated using the modified dilution method and respiratory quinone (RQ) analysis in mesotrophic Lake Biwa, Japan. The planktonic bacterial assemblages in the lake consisted of various RQ subgroups with different growth and mortality rates. The sum of total bacterial mortalities due to protistan grazing and viral lysis accounted for 96.6 % (range 89.0–107.2 %) of daily total bacterial production. This is the first report that successfully demonstrates a balanced relationship between bacterial production and losses using the modified dilution method in a lake. The growth rates of ubiquinone (UQ)-containing bacteria were faster than those of menaquinone-containing bacteria. Especially the dominant and fastest growing bacterial groups in the present study were the bacterial groups containing UQ-8 or UQ-10. The sum of their production and loss accounted for 60 % of carbon fluxes within the microbial loop. Thus, a large portion of the carbon cycling through the bacterial community in Lake Biwa can be explained by the carbon fluxes through dominant bacterial groups.     

Burkholderia cepacia Complex Phage-Antibiotic Synergy (PAS): Antibiotics Stimulate Lytic Phage Activity

The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing.