Lake Bacterial Communities in North Patagonian Andes: The Effect of the Nothofagus pumilio Treeline.

One of the most noticeable environmental discontinuities in mountains is the transition that exists in vegetation below and above the treeline. In the North Patagonian Andean lakes (between 900 and 1950 m a.s.l.), we analyzed the bacterial community composition of lakes in relation to surrounding vegetation (erected trees, krummholz belt, and bare rocks), dissolved organic carbon (DOC), and total dissolved nutrients (nitrogen, TDN and phosphorus, TDP). We observed a decrease in DOC, TDP, and TDN concentrations with altitude, reflecting shifts in the source inputs entering the lakes by runoff. Cluster analysis based on bacterial community composition showed a segregation of the lakes below treeline, from those located above. This first cluster was characterized by the cyanobacteria Cyanobium PCC-6307, while in the krummholz belt and bare rocks, bacterial communities were dominated by Actinobacteria hgcl-clade and Proteobacteria (Sandarakinorhabdus and Rhodovarius), with the presence of pigments such as actinorhodopsin, carotenoids, and bacteriochlorophyll a. The net relatedness index (NRI), which considers the community phylogenetic dispersion, showed that lakes located on bare rocks were structured by environmental filtering, while communities of lakes below treeline were structured by species interactions such as competition. Beta-diversity was higher among lakes below than among lakes located above the treeline. The contribution of species turnover was more important than nestedness. Our study brings light on how bacterial communities may respond to changes in the surrounding vegetation, highlighting the importance of evaluating different aspects of community structure to understand metacommunity organization.

Glacial Influence Affects Modularity in Bacterial Community Structure in Three Deep Andean North-Patagonian Lakes.

We analyze the bacteria community composition and the ecological processes structuring these communities in three deep lakes that receive meltwater from the glaciers of Mount Tronador (North-Patagonia, Argentina). Lakes differ in their glacial connectivity and in their turbidity due to glacial particles. Lake Ventisquero Negro is a recently formed proglacial lake and it is still in contact with the glacier. Lakes Mascardi and Frías lost their glacial connectivity during the Pleistocene-Holocene transition. Total dissolved solid concentration has a significant contribution to the environmental gradient determining the segregation of the three lakes. The newly formed lake Ventisquero Negro conformed a particular bacterial community that seemed to be more related to the microorganisms coming from glacier melting than to the other lakes of the basin. The net relatedness index (NRI) showed that the bacterial community of lake Ventisquero Negro is determined by environmental filtering, while in the other lakes, species interaction would be a more important driver. The co-occurrence network analysis showed an increase in modularity and in the number of modules when comparing Lake Ventisquero Negro with the two large glacier-fed lakes suggesting an increase in heterogeneity. At the same time, the presence of modules with phototrophic bacteria (Cyanobium strains) in lakes Frías and Mascardi would reflect the increase of this functional photosynthetic association. Overall, our results showed that the reduction in ice masses in Patagonia will affect downstream large deep Piedmont lakes losing the glacial influence in their bacterial communities.

A common temperature dependence of nutritional demands in ectotherms.

In light of ongoing climate change, it is increasingly important to know how nutritional requirements of ectotherms are affected by changing temperatures. Here, we analyse the wide thermal response of phosphorus (P) requirements via elemental gross growth efficiencies of Carbon (C) and P, and the Threshold Elemental Ratios in different aquatic invertebrate ectotherms: the freshwater model species Daphnia magna, the marine copepod Acartia tonsa, the marine heterotrophic dinoflagellate Oxyrrhis marina, and larvae of two populations of the marine crab Carcinus maenas. We show that they all share a non-linear cubic thermal response of nutrient requirements. Phosphorus requirements decrease from low to intermediate temperatures, increase at higher temperatures and decrease again when temperature is excessive. This common thermal response of nutrient requirements is of great importance if we aim to understand or even predict how ectotherm communities will react to global warming and nutrient-driven eutrophication.

Light dependence in the phototrophy-phagotrophy balance of constitutive and non-constitutive mixotrophic protists.

Mixotrophic protists display contrasting nutritional strategies and are key groups connecting planktonic food webs. They comprise constitutive mixotrophs (CMs) that have an innate photosynthetic ability and non-constitutive mixotrophs (NCMs) that acquire it from their prey. We modelled phototrophy and phagotrophy of two mixotrophic protists as a function of irradiance and prey abundance. We hypothesised that differences in their physiology (constitutive versus non-constitutive mixotrophy) can result in different responses to light gradients. We fitted the models with primary production and bacterivory data from laboratory and field experiments with the nanoflagellate Chrysochromulina parva (CM) and the ciliate Ophrydium naumanni (NCM) from north Andean Patagonian lakes. We found a non-monotonic response of phototrophy and phagotrophy to irradiance in both mixotrophs, which was successfully represented by our models. Maximum values for phototrophy and phagotrophy were found at intermediate irradiance coinciding with the light at the deep chlorophyll maxima in these lakes. At lower and higher irradiances, we found a decoupling between phototrophy and phagotrophy in the NCM while these functions were more coupled in the CM. Our modelling approach revealed the difference between both mixotrophic functional types on the balance between their nutritional strategies under different light scenarios. Thus, our proposed models can be applied to account how changing environmental conditions affect both primary and secondary production within the planktonic microbial food web.

Nutritional stress by means of high C:N ratios in the diet and starvation affects nitrogen isotope ratios and trophic fractionation of omnivorous copepods.

Nutritional stress, from feeding on low-quality diets or starvation, may cause changes in consumers' nitrogen isotope ratios (δ15N = 15N/14N) and trophic fractionation (∆15N = δ15Nconsumer - δ15Nfood source), however, research has shown mixed results in the magnitude and the direction of the change. This is potentially more complex for omnivores whose diets span a wide range of food resources. We conducted seasonal field samplings in Patagonian lakes and analyzed the relationship between seston (SES) quality parameters and the δ15N and ∆15N of an omnivorous copepod, Boeckella gracilipes (Bg). We also performed a 7-day laboratory starvation experiment, an extreme form of nutritional stress, to investigate if lack of food led to changes in δ15NBg values. Our field results showed that increasing values of the seston carbon to nitrogen ratio (C:NSES), chlorophyll a (Chl a), and δ15NSES were related to higher δ15NBg values. C:NSES and Chl a were also positively related to ∆15N; yet, C:NSES alone explained 70% of the variation. C:NSES values correlated with the presence of mixotrophic algae and ciliates that are key food resources for B. gracilipes. In our laboratory starvation experiment, the δ15NBg values increased significantly, pointing to use of internal N sources; yet, the change associated with starvation was less pronounced than that related to C:NSES changes in the field, suggesting depletion of the substrate pool in the former. We found that ∆15N values of omnivorous species consuming a low-quality diet would be higher than that from a conspecific with a high-quality diet; though fasting animals would show intermediate values.

Does the stoichiometric carbon:phosphorus knife edge apply for predaceous copepods?

Recent work has indicated that stoichiometric food quality in terms of the carbon:phosphorus (C:P) ratio affects consumers whether the imbalance involves a deficit or an excess of nutrients; hence, organisms exist on a "stoichiometric knife edge". While previous studies have focused primarily on autotroph-herbivore trophic transfer, nutritional imbalances may also affect the interactions between species at higher trophic levels. Since the foods of carnivores are normally stoichiometrically similar to the body compositions of those carnivores, they may be more severely affected than herbivores if imbalances become pronounced. We analysed the response of the predatory copepod Parabroteas sarsi to monospecific diet treatments consisting of high and low C:P prey items. These dietary treatments strongly affected the predator's elemental composition and growth, although prey selection, excretion, egestion, and respiration rates were not affected. We suggest that, due to their low threshold elemental ratio and a narrow C:P stoichiometric knife edge, these predators are highly vulnerable to stoichiometric imbalances, whether an excess or a deficit of nutrients is involved. Our results demonstrating this high sensitivity to prey C:P ratio show that the stoichiometric knife edge may apply to not only herbivores but also higher trophic levels. Thus, predators such as P. sarsi, with a much narrower range of food quality, may also be strongly affected by fluctuations in the quality of their prey, with negative consequences for their secondary production.

Shifts in food quality for herbivorous consumer growth: multiple golden means in the life history.

Consumer growth can be affected by imbalances between the nutrient content of the consumer and its food resource. Although ontogenetic-driven changes in animal composition are well documented, their potential consequences for the organism's sensitivity to food quality constraints have remained elusive. Here we show that the potential growth response of the copepod Mixodiaptomus laciniatus (as %RNA and RNA:DNA ratio) to the natural gradient of seston carbon (C) : nutrient ratio is unimodal and stage specific. Solution of the equation given by the first derivative function provided the optimum C : nutrient ratio for maximum stage-specific growth, which increased during ontogeny. The peakedness of the function indicated that animal vulnerability to suboptimal food quality decreased as juveniles reached adulthood. Consistent with these results, a field experiment demonstrated that potential consumer growth responded to variations in seston C: phosphorus ratio, and that early life stages were particularly vulnerable to suboptimal food quality.

Bacterial community structure in patagonian Andean Lakes above and below timberline: from community composition to community function.

Lakes located above the timberline are remote systems with a number of extreme environmental conditions, becoming physically harsh ecosystems, and sensors of global change. We analyze bacterial community composition and community-level physiological profiles in mountain lakes located in an altitude gradient in North Patagonian Andes below and above the timberline, together with dissolved organic carbon (DOC) characterization and consumption. Our results indicated a decrease in 71 % of DOC and 65 % in total dissolved phosphorus (TDP) concentration as well as in bacteria abundances along the altitude range (1,380 to 1,950 m a.s.l.). Dissolved organic matter (DOM) fluorescence analysis revealed a low global variability composed by two humic-like components (allochthonous substances) and a single protein-like component (autochthonous substances). Lakes below the timberline showed the presence of all the three components, while lakes above the timberline the protein-like compound constituted the main DOC component. Furthermore, bacterial community composition similarity and ordination analysis showed that altitude and resource concentration (DOC and TDP) were the main variables determining the ordination of groups. Community-level physiological profiles showed a mismatch with bacteria community composition (BCC), indicating the absence of a relationship between genetic and functional diversity in the altitude gradient. However, carbon utilization efficiencies varied according to the presence of different compounds in DOM bulk. The obtained results suggest that the different bacterial communities in these mountain lakes seem to have similar metabolic pathways in order to be able to exploit the available DOC molecules.

UV radiation simultaneously affects phototrophy and phagotrophy in nanoflagellate-dominated phytoplankton from an Andean shallow lake.

Mixotrophic nanoflagellates, that combine photosynthesis and phagotrophy, are important members of planktonic food webs in many aquatic environments depending on the balance among the different carbon and energy sources. We carried out field sampling and laboratory experiments with natural nanoflagellate assemblages from an Andean North-Patagonian lake exposing them or not to UVR, and measuring photosynthetic parameters and bacterivory. The effect of different light treatments on the photosynthetic efficiency was studied by the non-invasive, pulse amplitude-modulated (PAM) fluorescence technique, and bacterivory was assessed with fluorescently labeled bacteria (FLB). Mixotrophic nanoflagellates were clearly dominant (up to 90% of total phytoplankton and 88% of total nanoflagellate abundance), and in the experiments labeled bacteria were observed in more than 75% of mixotrophic cells. These results support the idea that these phytoflagellates were never entirely photosynthetic. The high light : phosphorus ratio and the high C : N : P ratio suggest a strong nutrient limitation towards P. Our results show that both functions, photosynthesis and bacteria ingestion, were simultaneously reduced by the same level of UVR. We estimated that UVR exposure of mixotrophic nanoflagellates reduced photosystem II activity between 23% and 31% while ingestion rates were reduced between 23% and 28%. Therefore, our results suggest that the different cell functions could be concurrently impacted by UVR, implying that patterns and rates of C transfer would be substantially altered in the microbial food web.

Short term fluctuating temperature alleviates Daphnia stoichiometric constraints.

In this study, we analysed how short term temperature fluctuation interacts with nutrient limitation in the vertical migrating Daphnia commutata. We hypothesize that short term (daily) temperature fluctuation will alleviate nutrient limitation. We carried out experiments analysing growth rates, phosphorus and RNA content of D. commutate grown under four different temperature regimes and two P-limited conditions. Our experiments showed that individuals grown under fluctuating temperature grew more than at the mean temperature. We estimated the expected sizes for the 15 °C treatment based on the Q10 and for the fluctuating temperature treatment. These expected sizes for both treatments resulted well below the observed ones. The P and RNA content of individuals grown at 10 °C were significantly higher than those at 20 °C, and when individuals grown at 10 °C were translocated to 20 °C they exerted an increased growth rate. Our results suggest that, under a regime of diel vertical migration, the temperature alternation would allow migrating organisms to alleviate the effect of severe nutrient limitation maintaining population growth. Under a scenario of global warming, where epilimnetic temperatures will increase, lake temperature will interact with nutrient limitation for consumers, but, organisms may be able to face these changes if they can still regularly move from a cold hypolimnion to a warmer epilimnion.

Global data set of long-term summertime vertical temperature profiles in 153 lakes.

Climate change and other anthropogenic stressors have led to long-term changes in the thermal structure, including surface temperatures, deepwater temperatures, and vertical thermal gradients, in many lakes around the world. Though many studies highlight warming of surface water temperatures in lakes worldwide, less is known about long-term trends in full vertical thermal structure and deepwater temperatures, which have been changing less consistently in both direction and magnitude. Here, we present a globally-expansive data set of summertime in-situ vertical temperature profiles from 153 lakes, with one time series beginning as early as 1894. We also compiled lake geographic, morphometric, and water quality variables that can influence vertical thermal structure through a variety of potential mechanisms in these lakes. These long-term time series of vertical temperature profiles and corresponding lake characteristics serve as valuable data to help understand changes and drivers of lake thermal structure in a time of rapid global and ecological change.

Effect of chronic UVR exposure on zooplankton molting and growth.

Molting is a crucial physiological process in arthropods development, growth, and adult reproduction, where the chitinolytic enzyme chitobiase (CB) and the apoptosis process (caspase-3 activity) play crucial roles. Both molecular endpoints have been observed to be affected by different toxics that may be present in aquatic environments. However, the role of ultraviolet radiation (UVR) in the molting process remains with poor evidence and the possible effect of the previous exposure on F1 generation is unknown. Here, we conducted laboratory experiments with chronic UVR exposure to test the effect on the molting process of Daphnia commutata. Our results showed a clear negative effect of the UVR that affected the molting process with a reduction in individual growth. This trend was also observed in CB and caspase-3 activities. Our results also suggest that the UV dose received by the mother and eggs has an additive effect with the dose received by the offspring. These results imply that the cumulative impact of small UVR doses (2 h per day, daily dose: 2520 J m-2 of 340 nm) on mothers and eggs (which cannot be discriminated in our experiments) can have an additive or synergistic effect along with the generations through a potential increase in lethal effect. Finally, the observed desynchronization in the molting process by UVR will affect the fitness of individuals and population dynamics.

Interactive effects of temperature, ultraviolet radiation and food quality on zooplankton alkaline phosphatase activity.

Ultraviolet Radiation (UVR) is a stressor for aquatic organisms affecting enzyme activities in planktonic populations because of the increase in reactive oxygen species. In addition, UVR exposure combined with other environmental factors (i.e. temperature and food quality) could have even higher detrimental effects. In this work, we aimed to determine the effect of UVR on somatic Alkaline Phosphatase Activity (APA) and Glutathione S-Transferase (GST) activity on the cladoceran Daphnia commutata under two different temperatures (10 °C and 20 °C) and under three food qualities (carbon:phosphorus ratios: 1150, 850 and 550). APA is a biomarker that is considered as a P deficiency indicator in zooplankton. Since recovery from UVR damage under dark conditions is an ATP depending reaction we also measured APA during recovery phases. We carried out a laboratory experiment combining different temperatures and food qualities with exposition to UVR followed by luminic and dark phases for recovery. In addition, we exposed organisms to H2O2, to establish if the response on APA to UVR was a consequence of the reactive oxygen species produced these short wavelengths. Our results showed that somatic APA was negatively affected by UVR exposure and this effect was enhanced under high temperature and low food quality. Consistently, GST activity was higher when exposed to UVR under both temperatures. The H2O2 experiments showed the same trend as UVR exposure, indicating that APA is affected mainly by oxidative stress than by direct effect of UVR on the enzyme. Finally, APA was affected in the dark phase of recovery confirming the P demands. These results enlighten the importance of food quality in the interacting effect of UVR and temperature, showing that C:P food ratio could determine the success or failure of zooplanktonic populations in a context of global change.

Low concentrations, potential ecological consequences: synthetic estrogens alter life-history and demographic structures of aquatic invertebrates.

Contraceptive drugs are nowadays found in aquatic environments around the globe. Particularly, 17α-ethinylestradiol (EE2) may act even at low concentrations, such as those recorded in natural ecosystems. We evaluated the physiological effects of EE2 on cyclopoids and calanoids, common copepods in both marine and freshwater communities. We used three EE2 concentrations and assessed its impact on activity of different physiological endpoints: Acetylcholinesterase (neurotransmission), Glutathione S-transferase (detoxifying system), and Caspase-3 (apoptosis). While EE2 exerts, distinctive effect on detoxifying and apoptotic systems, no effect on AChE was observed at environmental doses. Our results show that EE2 exposure affects differently copepod physiology endpoints, altering moulting process, adult recruitment in calanoids and calanoid to cyclopoid ratio. The ecological consequences of this underlying physiological process may affect since life history to population and community structures, and this represent a new aspects of this xenobiotic in natural systems.

Phylogenetic diversity of nonmarine picocyanobacteria.

We studied the phylogenetic diversity of nonmarine picocyanobacteria broadening the sequence data set with 43 new sequences of the 16S rRNA gene. The sequences were derived from monoclonal strains isolated from four volcanic high-altitude athalassohaline lakes in Mexico, five glacial ultraoligotrophic North Patagonian lakes and six Italian lakes of glacial, volcanic and morenic origin. The new sequences fall into a number of both novel and previously described clades within the phylogenetic tree of 16S rRNA gene. The new cluster of Lake Nahuel Huapi (North Patagonia) forms a sister clade to the subalpine cluster II and the marine Synechococcus subcluster 5.2. Our finding of the novel clade of 'halotolerants' close to the marine subcluster 5.3 (Synechococcus RCC307) constitutes an important demonstration that euryhaline and marine strains affiliate closely. The intriguing results obtained shed new light on the importance of the nonmarine halotolerants in the phylogenesis of picocyanobacteria.

Glacier melting and stoichiometric implications for lake community structure: zooplankton species distributions across a natural light gradient.

Glaciers around the globe are melting rapidly, threatening the receiving environments of the world's fresh water reservoirs with significant changes. The meltwater, carried by rivers, contains large amounts of suspended sediment particles, producing longitudinal gradients in the receiving lakes. These gradients may result in changes in the light : nutrient ratio that affect grazer performance by altering elemental food quality. Thus, glacial melting may induce a shift in the phytoplankton carbon : nutrient ratio and hence influence the dominance of herbivorous zooplankton through stoichiometric mechanisms. To test this hypothesis, we combined field and experimental data, taking advantage of a natural light intensity gradient caused by glacial clay input in a deep oligotrophic Patagonian lake. Across this gradient, we evaluated the abundances of two consumer taxa with different phosphorus requirements, the copepod Boeckella gracilipes and the cladoceran Daphnia commutata, using a six-station transect along the lake. We found significant differences in light : nutrient ratio and stoichiometric food quality of the seston, together with a switch from dominance of P-rich Daphnia in low carbon : nutrient stations to dominance of low-P copepods in high carbon : nutrient stations. The laboratory experiments confirmed that the difference in the carbon : nutrient ratio across the gradient is sufficient to impair Daphnia growth. The overall patterns are consistent with our prediction that shifts in the environmental light : nutrient ratio as a result of glacial melting would contribute to shifts in the dominance of stoichiometrically contrasting taxa in consumer guilds.

Rapid enzymatic response to compensate UV radiation in copepods.

Ultraviolet radiation (UVR) causes physical damage to DNA, carboxylation of proteins and peroxidation of lipids in copepod crustaceans, ubiquitous and abundant secondary producers in most aquatic ecosystems. Copepod adaptations for long duration exposures include changes in behaviour, changes in pigmentation and ultimately changes in morphology. Adaptations to short-term exposures are little studied. Here we show that short-duration exposure to UVR causes the freshwater calanoid copepod, Eudiaptomus gracilis, to rapidly activate production of enzymes that prevent widespread collateral peroxidation (glutathione S-transferase, GST), that regulate apoptosis cell death (Caspase-3, Casp-3), and that facilitate neurotransmissions (cholinesterase-ChE). None of these enzyme systems is alone sufficient, but they act in concert to reduce the stress level of the organism. The interplay among enzymatic responses provides useful information on how organisms respond to environmental stressors acting on short time scales.

Effect of ultraviolet radiation on acetylcholinesterase activity in freshwater copepods.

We analyzed the effects of UV radiation (UVR) effects on acetylcholinesterase (AChE) activity in two calanoid copepods, Boeckella gibbosa and Parabroteas sarsi that inhabit Patagonian shallow lakes. We studied the effect of experimental UVR (UV-B and UV-A) exposure on AChE activity in relation to basal antioxidant capacities of both copepods. Our experiments showed that UVR can effectively depress AChE activity, although with differences between species. In both copepods AChE was affected by UV-B, whereas UV-A only affected AChE in B. gibbosa. Both copepods also differed in body elemental composition (C:N:P), photoprotecting compound content (carotenoids and mycosporine-like amino acids) and enzymatic antioxidant capacity (glutathione S-transferase [GST]). Our results suggest that when exposed to UVR, AChE activity would depend more on the antioxidant capacity (GST) and P availability for enzyme synthesis than on the photoprotective compounds.

Ultraviolet radiation induces filamentation in bacterial assemblages from North Andean Patagonian lakes.

Through laboratory experiments, we tested whether UV radiation (UVR) induces filamentation in natural bacteria assemblages from North Andean Patagonian lakes. We incubated water from three different lakes for 72 h in four separate treatments: (1) UVR + PAR (photosynthetically active radiation), (2) 50% UVR + PAR, (3) PAR and (4) 50% PAR. The irradiance levels used in the experiments were equivalent to those registered at the epilimnion of the lakes. In the UVR treatments filamentation was induced after the first 24 h and the proportion continued to increase for the next 48-72 h. A comparison of the gross composition and diversity of the entire community (cells >0.2 microm) with bacterial filaments alone (>5.0 microm) showed that UVR-induced filamentation is not a feature of any particular cluster. By sequencing part of the 16S rRNA gene of the taxonomic units obtained using denaturing gels, we observed that strains in the beta-Proteobacteria group were of relatively high importance in filament formation, followed by Cytophaga-Flavobacterium-Bacteroides, gamma-Proteobacteria and alpha-Proteobacteria, whereas Actinobacteria were almost nonexistent in the filaments. We propose that UVR doses equivalent to those of Andean lakes produce bacterial morphological changes, and that all bacterial groups except Actinobacteria can potentially form filaments.