Phenotypic plasticity in freshwater picocyanobacteria.

Picocyanobacteria can occur as single-cell (Pcy) or as colonies (CPcy). Published evidence suggests that some Pcy strains have the capability to aggregate under certain culture conditions, however this has not been demonstrated to occur in natural environments. We investigated whether the Pcy and CPcy belong to the same species (i.e. phylotype), and the factors that determine their morphological and genetic variability in a hypertrophic shallow lake dominated by picocyanobacteria. Six main different morphologies and >30 phylotypes were observed. All sequences retrieved belonged to the 'Anathece + Cyanobium' clade (Synechococcales) that are known to have the capability of aggregation/disaggregation. The temporal variation of picocyanobacteria morphotype composition was weakly correlated with the DGGE temporal pattern, and could be explained by the composition of the zooplankton assemblage. Laboratory experiments confirmed that the small cladoceran Bosmina favoured the dominance of CPcy, i.e. Cyanodictyon doubled the size of the colonies when present, most likely through the aggregation of single-cell picocyanobacteria into colonies. Flow cytometry cell sorting and 16S rRNA + ITS sequencing of the Pcy and CPcy cytometrically-defined populations revealed that some phylotypes could be found in both sorted populations, suggesting phenotypic plasticity in which various Synechococcales phylotypes could be found in situ either as single-cells or as colonies.

Contrasting patterns of MAAs accumulation in two populations of the copepod Boeckella gracilipes.

The bio-accumulation of mycosporine-like amino acids (MAAs) is common in planktonic copepods that inhabit environments exposed to high levels of solar radiation. MAAs accumulation in copepods can be affected both by extrinsic (environmental) and intrinsic factors (local adaptation, genotype, etc.). Laboratory experiments were performed to study the bio-accumulation of MAAs in two geographically-isolated populations of Boeckella gracilipes from a mountain and a piedmont lake of North Patagonia. We performed two series of 10-day incubations of B. gracilipes from the different lakes applying two radiation conditions (PAR + UVR and darkness), at five different temperatures (5 to 20 °C) and providing a MAA-free flagellate as food. We assumed that differences in final MAAs concentrations between copepod populations should be exclusively due to environmental factors, and that any difference in the patterns of MAAs accumulation should exclusively arise from differences in MAAs concentration at the time of collection. MAAs concentration was three fold higher in B. gracilipes from Lake Verde than in copepods from the Lake Morenito. The MAAs suite was dominated (∼90%) by a combination of porphyra-334 and mycosporine-glycine in copepods from Lake Verde, and porphyra-334 and MAA-332 in those from Lake Morenito. Two exclusive MAA compounds were identified, mycosporine-glycine in copepods from Lake Verde and shinorine in the copepod population from Lake Morenito. Laboratory experiments showed that: (i) exposure to PAR + UVR stimulated the accumulation of MAAs in both copepod populations; (ii) temperature affected the response of MAAs and, remarkably, low temperatures stimulated MAAs accumulation even in dark incubations, (iii) the response to radiation and temperature in MAAs accumulation was more pronounced in the population with low initial MAAs than in the population with high initial MAAs concentrations. The differences in intrinsic factors between B. gracilipes populations, such as local adaptation to contrasting UV and temperature scenarios, among others, appear to play an important role in determining levels and patterns of MAAs accumulation in B. gracilipes.

Verrucomicrobiota are specialist consumers of sulfated methyl pentoses during diatom blooms.

Marine algae annually sequester petagrams of carbon dioxide into polysaccharides, which are a central metabolic fuel for marine carbon cycling. Diatom microalgae produce sulfated polysaccharides containing methyl pentoses that are challenging to degrade for bacteria compared to other monomers, implicating these sugars as a potential carbon sink. Free-living bacteria occurring in phytoplankton blooms that specialise on consuming microalgal sugars, containing fucose and rhamnose remain unknown. Here, genomic and proteomic data indicate that small, coccoid, free-living Verrucomicrobiota specialise in fucose and rhamnose consumption during spring algal blooms in the North Sea. Verrucomicrobiota cell abundance was coupled with the algae bloom onset and accounted for up to 8% of the bacterioplankton. Glycoside hydrolases, sulfatases, and bacterial microcompartments, critical proteins for the consumption of fucosylated and sulfated polysaccharides, were actively expressed during consecutive spring bloom events. These specialised pathways were assigned to novel and discrete candidate species of the Akkermansiaceae and Puniceicoccaceae families, which we here describe as Candidatus Mariakkermansia forsetii and Candidatus Fucivorax forsetii. Moreover, our results suggest specialised metabolic pathways could determine the fate of complex polysaccharides consumed during algae blooms. Thus the sequestration of phytoplankton organic matter via methyl pentose sugars likely depend on the activity of specialised Verrucomicrobiota populations.

New evidences of Roundup (glyphosate formulation) impact on the periphyton community and the water quality of freshwater ecosystems.

Argentina is the second largest world producer of soybeans (after the USA) and along with the increase in planted surface and production in the country, glyphosate consumption has grown in the same way. We investigated the effects of Roundup (glyphosate formulation) on the periphyton colonization. The experiment was carried out over 42 days in ten outdoor mesocosms of different typology: "clear" waters with aquatic macrophytes and/or metaphyton and "turbid" waters with great occurrence of phytoplankton or suspended inorganic matter. The herbicide was added at 8 mg L(-1) of the active ingredient (glyphosate) in five mesocosms while five were left as controls (without Roundup addition). The estimate of the dissipation rate (k) of glyphosate showed a half-life value of 4.2 days. Total phosphorus significantly increased in treated mesocosms due to Roundup degradation what favored eutrophication process. Roundup produced a clear delay in periphytic colonization in treated mesocosms and values of the periphytic mass variables (dry weight, ash-free dry weight and chlorophyll a) were always higher in control mesocosms. Despite the mortality of algae, mainly diatoms, cyanobacteria was favored in treated mesocosms. It was observed that glyphosate produced a long term shift in the typology of mesocosms, "clear" turning to "turbid", which is consistent with the regional trend in shallow lakes in the Pampa plain of Argentina. Based on our findings it is clear that agricultural practices that involve the use of herbicides such as Roundup affect non-target organisms and the water quality, modifying the structure and functionality of freshwater ecosystems.

Field evidence supports former experimental claims on the stimulatory effect of glyphosate on picocyanobacteria communities.

Glyphosate-based herbicides are the most commonly used herbicide worldwide. Although glyphosate is known to be toxic to aquatic organisms, it can also have stimulatory effects on small-size (ø <2 µm) cyanobacteria (Pcy) able to metabolize and degrade glyphosate and AMPA. Several previous experimental studies in micro- and mesocosms reported increases of Pcy abundance in response to glyphosate additions, but comparable field evidence is presently unavailable. We surveyed a large geographical area in order to collect information on Pcy abundance from lakes within the Pampa region (with over three decades of glyphosate usage) and lakes from Patagonia (with virtually no history of glyphosate usage). Fifty-two Pampean lakes and 24 Patagonian lakes were surveyed. We used three indicators of glyphosate impact: herbicide concentration, the presence of phosphonate metabolism genes (responsible for glyphosate and AMPA degradation) in environmental DNA samples, and descriptors of land use in the surrounding area of each lake. We addressed three questions: (1) is there field evidence of stimulatory effects of glyphosate on picocyanobacteria abundance? (2) is the magnitude of the effects of glyphosate in natural systems comparable to that reported under controlled experimental conditions? and (3), how do the effects of glyphosate compare to the effects of other potential environmental drivers of Pcy biomass? The collected evidence is consistent with the hypothesis that long-term agricultural practices relying on glyphosate-based technologies had important effects on freshwater microbial communities, particularly by promoting increases in picocyanobacteria abundance.

New findings on the effect of glyphosate on autotrophic and heterotrophic picoplankton structure: A microcosm approach.

Massive use of glyphosate-based herbicides in agricultural activities has led to the appearance of this herbicide in freshwater systems, which represents a potential threat to these systems and their communities. These herbicides can affect autotrophic and heterotrophic picoplankton abundance. However, little is known about glyphosate impact on the whole structure of these assemblages. Herein, we used an 8-day long microcosm approach under indoor controlled conditions to analyze changes in the structure of picoplankton exposed to a single pulse of glyphosate. The analyzed picoplankton correspond to two outdoor ponds with contrasting states: "clear" (chlorophyll-a = 3.48 µg L-1± 1.15; nephelometric turbidity, NTU = 1) and "turbid" (chlorophyll-a = 105.96 µg L-1 ± 15.3; NTU = 48). We evaluated herbicide impact on different picoplankton cytometric populations and further explored changes in bacterial dominant operational taxonomic units (OTUs) fingerprinting. We observed that glyphosate induced a drastic decrease in the abundance of phycocyanin-rich picocyanobacteria. Particularly, in the turbid system this effect resulted in an 85 % decrease in the abundance of the whole autotrophic picoplankton. Glyphosate also changed the structure of the heterotrophic fraction by means of changing bacterial dominant OTUs fingerprinting patterns in both systems and by shifting the relative abundances of cytometric groups in the clear scenario. These results demonstrate that upon glyphosate exposure picoplanktonic fractions face not only the already reported changes in abundance, but also alterations in the composition of cytometric groups and of bacterial dominant operational taxonomic units. This research provides suitable and still little explored tools to analyze agrochemical effects on picoplanktonic communities.

Alternative states drive the patterns in the bacterioplankton composition in shallow Pampean lakes (Argentina).

We assessed the influence of environmental factors in shaping the free-living bacterial community structure in a set of shallow lakes characterized by contrasting stable state patterns (clear-vegetated, inorganic-turbid and phytoplankton-turbid). Six temperate shallow lakes from the Pampa Plain (Argentina) were sampled over an annual cycle, and two fingerprinting techniques were applied: a 16S rDNA analysis was performed using denaturing gradient gel electrophoresis (DGGE) profiles, and a 16S-23S internally transcribed spacer region analysis was conducted by means of automated ribosomal intergenic spacer analysis (ARISA) profiles. Our results show that the steady state that characterized the different shallow lakes played a major role in structuring the community: the composition of free-living bacteria differed significantly between clear-vegetated, inorganic-turbid and phytoplankton-turbid shallow lakes. The state of the system was more important in determining these patterns than seasonality, geographical location or degree of hydrological connectivity. Moreover, this strong environmental control was particularly evident in the pattern observed in one of the lakes, which shifted from a clear to a turbid state over the course of the study. This lake showed a directional selection of species from a typical clear-like to a turbid-like community. The combined DGGE/ARISA approach revealed not only broad patterns among different alternative steady states, but also more subtle differences within different regimes.

Mycosporine-like amino acids in freshwater copepods: potential sources and some factors that affect their bioaccumulation.

Mycosporine-like amino acids (MAAs) are ubiquitous photoprotective compounds in aquatic environments. MAAs are synthesized by a wide variety of organisms (i.e. bacteria, fungi and algae) and their production is photoinducible by ultraviolet radiation (UVR) (280-400 nm) and/or photosynthetically active radiation (400-750 nm). Most animals however, are unable to synthesize MAAs and must acquire these compounds through their diet or from symbiotic organisms. In this paper, we investigate the possible sources of MAAs and factors (temperature and initial MAA concentration) that may affect their bioaccumulation in freshwater copepods. We found that MAA accumulation may occur even if the copepods are cultured on a MAA-free diet. In addition, we found that the bacteriostatic antibiotic, chloramphenicol, inhibits the bioaccumulation of MAAs. These two pieces of evidence suggest that the source of MAAs in these copepods may be prokaryotic organisms in close association with the animals. The two factors investigated in this study, temperature and initial MAA concentrations, were found to affect the rates at which MAAs are accumulated. Temperature had positive effects on both uptake and elimination rates. On the other hand, the rate of uptake decreased at the highest assayed initial MAA concentration, probably because the concentration of MAAs was already close to saturation.

Replicated mesocosm study on the role of natural ultraviolet radiation in high CDOM, shallow lakes.

The role of ultraviolet radiation on shallow, high CDOM (colored dissolved organic matter) lakes was investigated during two consecutive summers (1999 and 2000) in replicated mesocosms (rectangular fiberglass tanks). Each tank (volume: 300 L; depth: 40 cm) was covered with a layer (approximately 3 cm) of sediment from lake El Toro (40 degrees 14' S; 70 degrees 22' W) and filled with filtered water. The experimental design consisted of two treatments: full natural radiation (UV-exposed) and natural radiation without ultraviolet radiation (UV-shielded). UV-exposed and UV-shielded treatments differed in most studied variables as revealed by repeated measures ANOVA. UV-exposed tanks displayed lower CDOM levels (dissolved absorbance) of lower average molecular size (absorbance ratio between 250 and 365 nm), higher bacterial biomass, and lower chlorophyll a concentration. The effect on consumers (rotifers and crustaceans) was less noticeable. The results are consistent with UV stimulation of bacteria production mediated by higher rates of CDOM photobleaching, and the photoinhibition of planktonic algae. Thus, a major effect of UVR in shallow, high CDOM ecosystems appears to be the stimulation of heterotrophic pathways and a simultaneous inhibition of photoautotrophs.

Constitutive and UV-inducible synthesis of photoprotective compounds (carotenoids and mycosporines) by freshwater yeasts.

Twelve yeasts isolated from lakes of Northwestern Patagonia, Argentina, belonging to eight genera (Sporobolomyces, Sporidiobolus, Rhodotorula, Rhodosporidium, Cystofilobasidium, Cryptococcus, Torulaspora, and Candida) were analysed for their ability to produce photoprotective compounds. For this purpose, three laboratory experiments were performed to study the effect of photosynthetically active radiation (PAR) and PAR in combination with UV radiation (PAR + UVR) on the production of carotenoids and mycosporines. The synthesis of carotenoid compounds was clearly stimulated in six out of nine red yeast strains tested upon exposure to PAR or PAR + UVR; however, the latter conditions produced a stronger response than PAR alone. The increase in carotenoids in the red strains under PAR + UVR irradiation showed a negative exponential relationship with their basal carotenoid content, suggesting that cells with higher constitutive levels of carotenoids are less responsive to induction by PAR + UVR. Three red yeasts, Rhodotorula minuta, Rh. pinicola, and Rhodotorula sp., and the colourless Cryptococcus laurentii produced a UV-absorbing compound when exposed to PAR or PAR + UVR. This compound showed an absorption maximum at 309-310 nm and was identified as mycosporine-glutaminol-glucoside (myc-glu-glu). In these strains, exposure to PAR or PAR + UVR resulted in elevated concentrations of both carotenoids and myc-glu-glu. This is the first report on the production of mycosporines by yeasts. All strains that developed under PAR + UVR were able to synthesise carotenoids either constitutively or in response to PAR exposure, and a few of them also produced myc-glu-glu when exposed to PAR. Collectively, our results suggest that the presence of carotenoids, either alone or in combination with mycosporines, are required for sustaining growth under exposure to PAR + UVR in the freshwater yeast strains studied.