Microbial plankton responses to multiple environmental drivers in marine ecosystems with different phosphorus limitation degrees.

Multiple drivers are threatening the functioning of the microbial food webs and trophic interactions. Our understanding about how temperature, CO2, nutrient inputs, and solar ultraviolet radiation (UVR) availability interact to alter ecosystem functioning is scarce because research has focused on single and double interactions. Moreover, the role that the degree of in situ nutrient limitation could play in the outcome of these interactions has been largely neglected, despite it is predominant in marine ecosystems. We address these uncertainties by combining remote-sensing analyses, and a collapsed experimental design with natural microbial communities from Mediterranean Sea and Atlantic Ocean exposed to temperature, nutrients, CO2, and UVR interactions. At the decade scale, we found that more intense and frequent (and longer lasting) Saharan dust inputs (and marine heatwaves) were only coupled with reduced phytoplankton biomass production. When microbial communities were concurrently exposed to future temperature, CO2, nutrient, and UVR conditions (i.e. the drivers studied over long-term scales), we found shifts from net autotrophy [primary production:respiration (PP:R) ratio > 1] towards a metabolic equilibrium (PP:R ratio ~ 1) or even a net heterotrophy (PP:R ratio < 1), as P-limitation degree was higher (i.e. Atlantic Ocean). These changes in the metabolic balance were coupled with a weakened phytoplankton-bacteria interaction (i.e. bacterial carbon demand exceeded phytoplankton carbon supply. Our work reveals that an accentuated in situ P limitation may promote reductions both in carbon uptake and fluxes between trophic levels in microbial plankton communities under global-change conditions. We show that considering long-term series can aid in identifying major local environmental drivers (i.e. temperature and nutrients in our case), easing the design of future global-change studies, but also that the abiotic environment to which microbial plankton communities are acclimated should be taken into account to avoid biased predictions concerning the effects of multiple interacting global-change drivers on marine ecosystems.

Regulation of Phagotrophy by Prey, Low Nutrients, and Low Light in the Mixotrophic Haptophyte Isochrysis galbana.

Mixotrophy combines autotrophy and phagotrophy in the same cell. However, it is not known to what extent the phagotrophy influences metabolism, cell composition, and growth. In this work, we assess, on the one hand (first test), the role of phagotrophy on the elemental and biochemical composition, cell metabolism, and enzymes related to C, N, and S metabolism of Isochrysis galbana Parke, 1949. On the other hand, we study how a predicted increase of phagotrophy under environmental conditions of low nutrients (second test) and low light (third test) can affect its metabolism and growth. Our results for the first test revealed that bacterivory increased the phosphorous and iron content per cell, accelerating cell division and improving the cell fitness; in addition, the stimulation of some C and N enzymatic routes help to maintain, to some degree, compositional homeostasis. Under nutrient or light scarcity, I. galbana grew more slowly despite greater bacterial consumption, and the activities of key enzymes involved in C, N, and S metabolism changed according to a predominantly phototrophic strategy of nutrition in this alga. Contrary to recent studies, the stimulation of phagotrophy under low nutrient and low irradiance did not imply greater and more efficient C flux.

Uncoupled phytoplankton-bacterioplankton relationship by multiple drivers interacting at different temporal scales in a high-mountain Mediterranean lake.

Global-change stressors act under different timing, implying complexity and uncertainty in the study of interactive effects of multiple factors on planktonic communities. We manipulated three types of stressors acting in different time frames in an in situ experiment: ultraviolet radiation (UVR); phosphorus (P) concentration; temperature (T) in an oligotrophic Mediterranean high-mountain lake. The aim was to examine how the sensitivity of phytoplankton and bacterioplankton to UVR and their trophic relationship change under nutrient acclimation and abrupt temperature shifts. Phytoplankton and bacteria showed a common pattern of metabolic response to UVR × P addition interaction, with an increase in their production rates, although evidencing an inhibitory UVR effect on primary production (PP) but stimulatory on bacterial production (HBP). An abrupt T shift in plankton acclimated to UVR and P addition decreased the values of PP, evidencing an inhibitory UVR effect, whereas warming increased HBP and eliminated the UVR effect. The weakening of commensalistic and predatory relationship between phyto- and bacterioplankton under all experimental conditions denotes the negative effects of present and future global-change conditions on planktonic food webs towards impairing C flux within the microbial loop.

Interplay between resistance and resilience governs the stability of a freshwater microbial food web under multiple stressors.

Energy (photosynthetically active [PAR] and ultraviolet [UVR] radiation) and matter (organic and inorganic nutrients) fluxes regulate the ecosystem's stability. However, the mechanisms underpinning the potential interplay between resistance and resilience to shifts in nutrient inputs and UVR are poorly understood. To assess how the UVR × nutrients interaction alters ecosystem stability, we exposed in situ a microbial food web from an oligotrophic ecosystem to: (1) two light (UVR + PAR and PAR), and (2) four nutrient (ambient concentrations, phosphorus [P], carbon [C] and C × P addition) treatments for three weeks. During this period, we quantified the community composition and biomass, sestonic P and C:P ratio, primary [PP] and bacterial [BP] production, community [CR] and bacterial [BR] respiration, excreted organic carbon [EOC], as well as the commensalistic phytoplankton-bacteria interaction (i.e. bacterial carbon demand [BCD]:EOC ratio) and the metabolic balance of the ecosystem (i.e. [PP:R] ratio). The stability of all response variables under the four environmental scenarios tested (i.e. UVR, UVR × C, UVR × P, and UVR × C × P) was quantified by means of the resistance and resilience indexes. The microbial community was dominated by phototrophs during the experimental period regardless of the treatment considered. The most complex scenario, i.e. UVR × C × P, decreased the resistance for all variables, except for BR and the PP:R ratio. Despite that PP:R ratio showed the highest resistance under such scenario, it was >1 in all environmental scenarios (i.e. net autotrophic), except under the UVR × C interaction, where, concomitant with increased resilience, the balance shifted towards net heterotrophy (PP:R < 1). Under the UVR × C × P scenario, the metabolic balance of the ecosystem proved strongly resistant due mainly to high resistance of bacterial respiration and a firm stability of the commensalistic interaction. Our results evidence that the high resilience of phototrophs (favoring their predominance over mixo- and heterotrophs) may lead to the maintenance of the autotrophic nature and carbon (C) sink capacity of the ecosystem.

Climate-driven shifts in algal-bacterial interaction of high-mountain lakes in two years spanning a decade.

Algal-bacterial interactions include mutualism, commensalism, and predation. However, how multiple environmental conditions that regulate the strength and prevalence of a given interaction remains unclear. Here, we test the hypothesis that the prevailing algal-bacterial interaction shifted in two years (2005 versus 2015), due to increased temperature (T) and Saharan dust depositions in high-mountain lakes of Sierra Nevada (S Spain). Our results support the starting hypothesis that the nature of the prevailing algal-bacterial interaction shifted from a bacterivory control exerted by algae to commensalism, coinciding with a higher air and water T as well as the lower ratio sestonic nitrogen (N): phosphorous (P), related to greater aerosol inputs. Projected global change conditions in Mediterranean region could decline the functional diversity and alter the role of mixotrophy as a carbon (C) by-pass in the microbial food web, reducing the biomass-transfer efficiency up the web by increasing the number of trophic links.

Quantification of carbon and phosphorus co-limitation in bacterioplankton: new insights on an old topic.

Because the nature of the main resource that limits bacterioplankton (e.g. organic carbon [C] or phosphorus [P]) has biogeochemical implications concerning organic C accumulation in freshwater ecosystems, empirical knowledge is needed concerning how bacteria respond to these two resources, available alone or together. We performed field experiments of resource manipulation (2×2 factorial design, with the addition of C, P, or both combined) in two Mediterranean freshwater ecosystems with contrasting trophic states (oligotrophy vs. eutrophy) and trophic natures (autotrophy vs. heterotrophy, measured as gross primary production:respiration ratio). Overall, the two resources synergistically co-limited bacterioplankton, i.e. the magnitude of the response of bacterial production and abundance to the two resources combined was higher than the additive response in both ecosystems. However, bacteria also responded positively to single P and C additions in the eutrophic ecosystem, but not to single C in the oligotrophic one, consistent with the value of the ratio between bacterial C demand and algal C supply. Accordingly, the trophic nature rather than the trophic state of the ecosystems proves to be a key feature determining the expected types of resource co-limitation of bacteria, as summarized in a proposed theoretical framework. The actual types of co-limitation shifted over time and partially deviated (a lesser degree of synergism) from the theoretical expectations, particularly in the eutrophic ecosystem. These deviations may be explained by extrinsic ecological forces to physiological limitations of bacteria, such as predation, whose role in our experiments is supported by the relationship between the dynamics of bacteria and bacterivores tested by SEMs (structural equation models). Our study, in line with the increasingly recognized role of freshwater ecosystems in the global C cycle, suggests that further attention should be focussed on the biotic interactions that modulate resource co-limitation of bacteria.

Nucleic acid content in crustacean zooplankton: bridging metabolic and stoichiometric predictions.

Metabolic and stoichiometric theories of ecology have provided broad complementary principles to understand ecosystem processes across different levels of biological organization. We tested several of their cornerstone hypotheses by measuring the nucleic acid (NA) and phosphorus (P) content of crustacean zooplankton species in 22 high mountain lakes (Sierra Nevada and the Pyrenees mountains, Spain). The P-allocation hypothesis (PAH) proposes that the genome size is smaller in cladocerans than in copepods as a result of selection for fast growth towards P-allocation from DNA to RNA under P limitation. Consistent with the PAH, the RNA:DNA ratio was >8-fold higher in cladocerans than in copepods, although 'fast-growth' cladocerans did not always exhibit higher RNA and lower DNA contents in comparison to 'slow-growth' copepods. We also showed strong associations among growth rate, RNA, and total P content supporting the growth rate hypothesis, which predicts that fast-growing organisms have high P content because of the preferential allocation to P-rich ribosomal RNA. In addition, we found that ontogenetic variability in NA content of the copepod Mixodiaptomus laciniatus (intra- and interstage variability) was comparable to the interspecific variability across other zooplankton species. Further, according to the metabolic theory of ecology, temperature should enhance growth rate and hence RNA demands. RNA content in zooplankton was correlated with temperature, but the relationships were nutrient-dependent, with a positive correlation in nutrient-rich ecosystems and a negative one in those with scarce nutrients. Overall our results illustrate the mechanistic connections among organismal NA content, growth rate, nutrients and temperature, contributing to the conceptual unification of metabolic and stoichiometric theories.

Maximum in the middle: nonlinear response of microbial plankton to ultraviolet radiation and phosphorus.

The responses of heterotrophic microbial food webs (HMFW) to the joint action of abiotic stressors related to global change have been studied in an oligotrophic high-mountain lake. A 2×5 factorial design field experiment performed with large mesocosms for >2 months was used to quantify the dynamics of the entire HMFW (bacteria, heterotrophic nanoflagellates, ciliates, and viruses) after an experimental P-enrichment gradient which approximated or surpassed current atmospheric P pulses in the presence vs. absence of ultraviolet radiation. HMFW underwent a mid-term (<20 days) acute development following a noticeable unimodal response to P enrichment, which peaked at intermediate P-enrichment levels and, unexpectedly, was more accentuated under ultraviolet radiation. However, after depletion of dissolved inorganic P, the HMFW collapsed and was outcompeted by a low-diversity autotrophic compartment, which constrained the development of HMFW and caused a significant loss of functional biodiversity. The dynamics and relationships among variables, and the response patterns found, suggest the importance of biotic interactions (predation/parasitism and competition) in restricting HMFW development, in contrast to the role of abiotic factors as main drivers of autotrophic compartment. The response of HMFW may contribute to ecosystem resilience by favoring the maintenance of the peculiar paths of energy and nutrient-mobilization in these pristine ecosystems, which are vulnerable to threats by the joint action of abiotic stressors related to global change.

Effects of ultraviolet radiation and nutrients on the structure-function of phytoplankton in a high mountain lake.

The combined effect of high solar ultraviolet radiation (UVR) and nutrient supply in a phytoplankton community of a high mountain lake is analyzed in a in situ experiment for 6 days with 2 × 2 factorial design. Interactive UVR × nutrient effects on structural and functional variables (algal biomass, chlorophyll a (chl a), primary production (PP), maximal electron transport rate (ETR(max)), and alkaline phosphatase activity (APA)), as well as stoichiometric ones (sestonic N per cell and N:P ratio) were found. Under non-nutrient enriched conditions, no deleterious effects of UVR on structural variables, PP, photosynthetic efficiency and ETR(max) were observed, whereas only particulate and total APA were affected by UVR. However, percentage excreted organic carbon (%EOC), dissolved APA and sestonic C and P per cell increased under UVR, leading to a decrease in algal C:P and N:P ratios. After nutrient enrichment, chl a, total algal biomass and PP were negatively affected by UVR whereas %EOC, ETR(max) and internal C, P and N content increased. We suggest that the mechanism of algal acclimation to UVR in this high UVR flux ecosystem seems to be related to the increase of internal algal P-content mediated by physiological mechanisms to save P and by a stimulatory UVR effect on dissolved extracellular APA. The mechanism involved in the unmasking effect of UVR after nutrient-enrichment may be the result of a greater sensitivity to UVR-induced cell damage, making the negative UVR effects more evident.

Patterns of resource limitation of bacteria along a trophic gradient in Mediterranean inland waters.

The nature of the resource that limits heterotrophic bacteria, i.e. mineral nutrients or carbon (C), has consequences for biogeochemical cycles in aquatic ecosystems. Our aim was to identify the resource [C or phosphorus (P)] that mainly limits bacteria in a set of 31 Mediterranean inland water ecosystems spanning a wide trophic range. We followed an intersystem observational approach with three complementary perspectives, comparing the bacterial demand with the resource supply in terms of both the quantity (demand : supply ratio for C and P) and quality (C : P ratio of demand and supply), and assessing the relative strength of each resource in controlling bacterial production. The trophic gradient revealed a shift in the main limiting resource for bacteria, from C at the oligotrophic end (typically high-mountain, low-productivity lakes) to mainly P at the eutrophic end (typically nonmountain, high-productivity lakes). The patterns of resource limitation of bacteria found here may be related to the autotrophic nature of most of the studied ecosystems linked to a Mediterranean climate regime as representative of lakes with low inputs of allocthonous C. These patterns are consistent with the theoretical approaches and may potentially shape the contribution of this type of ecosystems to biogeochemical cycles.