An inquiline mosquito modulates microbial diversity and function in an aquatic microecosystem.

Understanding microbial roles in ecosystem function requires integrating microscopic processes into food webs. The carnivorous pitcher plant, Sarracenia purpurea, offers a tractable study system where diverse food webs of macroinvertebrates and microbes facilitate digestion of captured insect prey, releasing nutrients supporting the food web and host plant. However, how interactions between these macroinvertebrate and microbial communities contribute to ecosystem functions remains unclear. We examined the role of the pitcher plant mosquito, Wyeomyia smithii, in top-down control of the composition and function of pitcher plant microbial communities. Mosquito larval abundance was enriched or depleted across a natural population of S. purpurea pitchers over a 74-day field experiment. Bacterial community composition and microbial community function were characterized by 16S rRNA amplicon sequencing and profiling of carbon substrate use, bulk metabolic rate, hydrolytic enzyme activity, and macronutrient pools. Bacterial communities changed from pitcher opening to maturation, but larvae exerted minor effects on high-level taxonomic composition. Higher larval abundance was associated with lower diversity communities with distinct functions and elevated nitrogen availability. Treatment-independent clustering also supported roles for larvae in curating pitcher microbial communities through shifts in community diversity and function. These results demonstrate top-down control of microbial functions in an aquatic microecosystem.

The ongoing need for rates: can physiology and omics come together to co-design the measurements needed to understand complex ocean biogeochemistry?

The necessity to understand the influence of global ocean change on biota has exposed wide-ranging gaps in our knowledge of the fundamental principles that underpin marine life. Concurrently, physiological research has stagnated, in part driven by the advent and rapid evolution of molecular biological techniques, such that they now influence all lines of enquiry in biological oceanography. This dominance has led to an implicit assumption that physiology is outmoded, and advocacy that ecological and biogeochemical models can be directly informed by omics. However, the main modeling currencies are biological rates and biogeochemical fluxes. Here, we ask: how do we translate the wealth of information on physiological potential from omics-based studies to quantifiable physiological rates and, ultimately, to biogeochemical fluxes? Based on the trajectory of the state-of-the-art in biomedical sciences, along with case-studies from ocean sciences, we conclude that it is unlikely that omics can provide such rates in the coming decade. Thus, while physiological rates will continue to be central to providing projections of global change biology, we must revisit the metrics we rely upon. We advocate for the co-design of a new generation of rate measurements that better link the benefits of omics and physiology.

Growth parameters and responses of green algae across a gradient of phototrophic, mixotrophic and heterotrophic conditions.

Many studies have shown that algal growth is enhanced by organic carbon and algal mixotrophy is relevant for physiology and commercial cultivation. Most studies have tested only a single organic carbon concentration and report different growth parameters which hampers comparisons and improvements to algal cultivation methodology. This study compared growth of green algae Chlorella vulgaris and Chlamydomonas reinhardtii across a gradient of photoautotrophic-mixotrophic-heterotrophic culture conditions, with five acetate concentrations. Culture growth rates and biomass achieved were compared using different methods of biomass estimation. Both species grew faster and produced the most biomass when supplied with moderate acetate concentrations (1-4 g L-1), but light was required to optimize growth rates, biomass yield, cell size and cell chlorophyll content. Higher acetate concentration (10 g L-1) inhibited algal production. The choice of growth parameter and method to estimate biomass (optical density (OD), chlorophyll a fluorescence, flow cytometry, cell counts) affected apparent responses to organic carbon, but use of OD at 600, 680 or 750 nm was consistent. There were apparent trade-offs among exponential growth rate, maximum biomass, and culture time spent in exponential phase. Different cell responses over 1-10 g L-1 acetate highlight profound physiological acclimation across a gradient of mixotrophy. In both species, cell size vs cell chlorophyll relationships were more constrained in photoautotrophic and heterotrophic cultures, but under mixotrophy, and outside exponential growth phase, these relationships were more variable. This study provides insights into algal physiological responses to mixotrophy but also has practical implications for choosing parameters for monitoring commercial algal cultivation.

Bacterial Recruitment to Carnivorous Pitcher Plant Communities: Identifying Sources Influencing Plant Microbiome Composition and Function.

Processes influencing recruitment of diverse bacteria to plant microbiomes remain poorly understood. In the carnivorous pitcher plant Sarracenia purpurea model system, individual pitchers open to collect rainwater, invertebrates and a diverse microbial community, and this detrital food web is sustained by captured insect prey. This study examined how potential sources of bacteria affect the development of the bacterial community within pitchers, how the host plant tissue affects community development and how established vs. assembling communities differ. In a controlled greenhouse experiment, seven replicate pitchers were allocated to five treatments to exclude specific bacterial sources or host tissue: milliQ water only, milliQ + insect prey, rainwater + prey, established communities + prey, artificial pitchers with milliQ + prey. Community composition and functions were examined over 8-40 weeks using bacterial gene sequencing and functional predictions, measurements of cell abundance, hydrolytic enzyme activity and nutrient transformations. Distinct community composition and functional differences between artificial and real pitchers confirm an important influence of host plant tissue on community development, but also suggest this could be partially related to host nutrient uptake. Significant recruitment of bacteria to pitchers from air was evident from many taxa common to all treatments, overlap in composition between milliQ, milliQ + prey, and rainwater + prey treatments, and few taxa unique to milliQ only pitchers. Community functions measured as hydrolytic enzyme (chitinase, protease) activity suggested a strong influence of insect prey additions and were linked to rapid transformation of insect nutrients into dissolved and inorganic sources. Bacterial taxa found in 6 of 7 replicate pitchers within treatments, the "core microbiome" showed tighter successional trajectories over 8 weeks than all taxa. Established pitcher community composition was more stable over 8 weeks, suggesting a diversity-stability relationship and effect of microinvertebrates on bacteria. This study broadly demonstrates that bacterial composition in host pitcher plants is related to both stochastic and specific bacterial recruitment and host plants influence microbial selection and support microbiomes through capture of insect prey.

Exploring Microbiome Functional Dynamics through Space and Time with Trait-Based Theory.

Microbiomes play essential roles in the health and function of animal and plant hosts and drive nutrient cycling across ecosystems. Integrating novel trait-based approaches with ecological theory can facilitate the prediction of microbial functional traits important for ecosystem functioning and health. In particular, the yield-acquisition-stress (Y-A-S) framework considers dominant microbial life history strategies across gradients of resource availability and stress. However, microbiomes are dynamic, and spatial and temporal shifts in taxonomic and trait composition can affect ecosystem functions. We posit that extending the Y-A-S framework to microbiomes during succession and across biogeographic gradients can lead to generalizable rules for how microbiomes and their functions respond to resources and stress across space, time, and diverse ecosystems. We demonstrate the potential of this framework by applying it to the microbiomes hosted by the carnivorous pitcher plant Sarracenia purpurea, which have clear successional trajectories and are distributed across a broad climatic gradient.

Role of nearshore benthic algae in the Lake Michigan silica cycle.

Si cycling is linked with processes from global carbon sequestration to community composition and is especially important in aquatic ecosystems. Lake Michigan has seen dramatic fluctuations in dissolved silica (dSi) over several decades, which have been examined in the context of planktonic processes (diatom blooms), but the role of benthic organisms (macroalgae and their epiphytes) in Si cycling have not been explored. To assess significance of nearshore benthic algae in Si dynamics, we assembled dSi data from an offshore site sampled since the late 1980's, and sampled off three Milwaukee beaches during 2005-19. Using colorimetric assays and alkaline digestion, we measured dSi, biogenic silica in particulate suspended material (pSi) and biogenic silica in benthic macroalgae (Cladophora) and epiphytic diatoms (bSi). Offshore, dSi increased about 1 µM per year from 25 µM in the late 1980's to nearly 40 µM in 2019. Nearshore dSi fluctuated dramatically annually, from near zero to concentrations similar to offshore. Both Cladophora and its epiphytes contained significant bSi, reaching up to 30% of dry mass (300 mg Si g dry mass-1) of the assemblage in summer. Microscopic analyses including localization with a Si-specific-stain and X-ray microanalysis showed bSi in epiphytic diatom cells walls, but the nature and localization of Si in macroalgae remained unclear. A simple model was developed estimating Si demand of algae using the areal macroalgal biomass, growth rates inferred from P-content, and bSi content, and comparing Si demand with dSi available in the water column. This indicated that 7-70% of the dSi in water overlying nearshore benthic algal beds could be removed per day. Key elements of the Si cycle, including which organisms sequester bSi and how rapidly Si is recycled, remain unclear. This work has implications for coastal marine waters where large macroalgal biomass accumulates but bSi content is virtually unknown.

Toward enhanced performance of integrated photo-bioelectrochemical systems: Taxa and functions in bacteria-algae communities.

An integrated photo-bioelectrochemical (IPB) system uses microalgae in the cathode of a microbial fuel cell to achieve higher electricity generation and nutrient removal from wastewater. Using multivariate analysis and surveys of IPB studies, this paper identifies key algal and bacterial taxa and discusses their functions critical for IPB performance. Unicellular algae with high photosynthetic oxygen production and biofilm formation can enhance IPB energy production. Diverse bacterial taxa achieve nitrogen transformations and can improve total nitrogen removal. Understanding bacteria-algae interactions via quorum sensing in the IPB cathode may potentially aid in boosting system performance. Future advances in development of IPBs for wastewater treatment will benefit from interdisciplinary collaboration in analysis of microbial community functions.

Effects of bacterial inoculation and nitrogen loading on bacterial-algal consortium composition and functions in an integrated photobioelectrochemical system.

An integrated photo-bioelectrochemical system (IPB) for wastewater treatment combines a microbial fuel cell with an algal bioreactor, eliminating requirements for aeration, promoting electricity generation, remediating nutrients and producing algal biomass for conversion into biofuel or other bioproducts. To examine strategies for improving IPB functions of electrochemical output and nutrient removal efficiency, this study tested effects of cathode bacterial inoculation and nitrogen loading on cathode microbial community and IPB performance. IPB cathodes were inoculated with the green alga Chlorella vulgaris, in combination with nitrite-oxidizing bacteria (NOB) Nitrobacter winogradskyi, and/or ammonium-oxidizing bacteria (AOB) Nitrosomonas europaea. IPB performance was examined before and after nitrifying bacteria inoculations and under three ammonium loading concentrations in the wastewater medium. Bacterial communities in the cathode suspension and biofilm were examined by 16S rRNA gene sequence analysis. Relative to the algae only control, cathode inoculation with NOB and/or AOB improved net nutrient removal, but resulted in reduced dissolved oxygen availability, which impaired electricity generation. Higher ammonium loading increased electricity production and nutrient removal, possibly by overcoming algal-bacterial competition. Inoculation with nitrifying bacteria resulted in minor changes to total bacterial composition and AOB or NOB comprised <3% of total sequences after 1 month. Community composition changed more dramatically following increase in ammonium-N concentration from 40 to 80 mg L-1. Manipulation of N loading could be a useful strategy to improve IPB performance, while inoculation of AOB or NOB may be beneficial for treatment of water with high ammonium loading when N removal is the primary system goal.

Diverse microbial communities hosted by the model carnivorous pitcher plant Sarracenia purpurea: analysis of both bacterial and eukaryotic composition across distinct host plant populations.

BACKGROUND: The pitcher plant Sarracenia purpurea supplements nutrient acquisition through carnivory, capturing insect prey which are digested by a food web community of eukaryotes and bacteria. While the food web invertebrates are well studied, and some recent studies have characterized bacteria, detailed genetic analysis of eukaryotic diversity is lacking. This study aimed to compare eukaryotic and bacterial composition and diversity of pitcher communities within and between populations of host plants in nearby but distinct wetland habitats, and to characterize microbial functions across populations and in comparison with another freshwater community. METHODS: Pitcher fluid was sampled from the two wetlands, Cedarburg and Sapa Bogs, community DNA was extracted, and 16S and 18S rRNA amplicons were sequenced and data processed for community-level comparisons. RESULTS AND CONCLUSIONS: Bacterial diversity in the small pitcher volume rivaled that of larger aquatic communities. Between pitcher plant populations, several bacterial families (Kiloniellaceae, Acetobacteraceae, Xanthobacteraceae, Sanguibacteraceae, Oligoflexaceae, Nitrosomonadaceae, Chromatiaceae, Saprospiraceae) were significantly higher in one population. However, although predicted pitcher bacterial functions were distinct from other freshwater communities, especially for some amino acid metabolism, functions were similar across all the pitchers in the two populations. This suggests some functional redundancy among bacterial taxa, and that functions converge to achieve similar food web processes. The sequencing identified a previously under-appreciated high diversity of ciliates, Acari mites, fungi and flagellates in pitcher communities; the most abundant sequences from eukaryotic taxa were Oligohymenophorea ciliates, millipedes and Ichthyosporea flagellates. Two thirds of taxa were identified as food web inhabitants and less than one third as prey organisms. Although eukaryotic composition was not significantly different between populations, there were different species of core taxonomic groups present in different pitchers-these differences may be driven by wetland habitats providing different populations to colonize new pitchers. Eukaryotic composition was more variable than bacterial composition, and there was a poor relationship between bacterial and eukaryotic composition within individual pitchers, suggesting that colonization by eukaryotes may be more stochastic than for bacteria, and bacterial recruitment to pitchers may involve factors other than prey capture and colonization by eukaryotic food web inhabitants.

Regulation of Hydrolytic Enzyme Activity in Aquatic Microbial Communities Hosted by Carnivorous Pitcher Plants.

Carnivorous pitcher plants Sarracenia purpurea host diverse eukaryotic and bacterial communities which aid in insect prey digestion, but little is known about the functional processes mediated by the microbial communities. This study aimed to connect pitcher community diversity with functional nutrient transformation processes, identifying bacterial taxa, and measuring regulation of hydrolytic enzyme activity in response to prey and alternative nutrient sources. Genetic analysis identified diverse bacterial taxa known to produce hydrolytic enzyme activities. Chitinase, protease, and phosphatase activities were measured using fluorometric assays. Enzyme activity in field pitchers was positively correlated with bacterial abundance, and activity was suppressed by antibiotics suggesting predominantly bacterial sources of chitinase and protease activity. Fungi, algae, and rotifers observed could also contribute enzyme activity, but fresh insect prey released minimal chitinase activity. Activity of chitinase and proteases was upregulated in response to insect additions, and phosphatase activity was suppressed by phosphate additions. Particulate organic P in prey was broken down, appearing as increasing dissolved organic and inorganic P pools within 14 days. Chitinase and protease were not significantly suppressed by availability of dissolved organic substrates, though organic C and N stimulated bacterial growth, resulting in elevated enzyme activity. This comprehensive field and experimental study show that pitcher plant microbial communities dynamically regulate hydrolytic enzyme activity, to digest prey nutrients to simpler forms, mediating biogeochemical nutrient transformations and release of nutrients for microbial and host plant uptake.

Invasive dreissenid mussels and benthic algae in Lake Michigan: characterizing effects on sediment bacterial communities.

Dreissenid mussels have invaded the Laurentian Great Lakes causing dramatic changes to benthic-pelagic interactions. Despite research on food web impacts, there is limited data on mussel effects on benthic bacterial communities. This study examined effects of dreissenid mussels and benthic algae on sediment bacterial community composition and diversity. Triplicate experimental sediment plus lake water microcosms were used and either mussels, benthic algae or both were added. Changes in water nutrient chemistry and sediment bacterial communities were monitored using 16S rRNA amplicon sequencing, over 21 days. When mussels were present, nitrate and soluble reactive P increased significantly as the dominant N and P forms. Bacterial diversity increased in all microcosms, although bacterial community composition was distinct between treatment. Higher nitrate in mussel microcosms was accompanied by increases in nitrifying taxa (Nitrospira, Nitrosomonas), which are important in oxidizing mussel-excreted ammonium. Microcosms with algal additions showed increases in bacterial taxa capable of degrading algal cellulose, and Pelagibacter (SAR11) disappeared from all but control microcosms. This study suggests that bacterial communities in lake sediments respond to mussels and algae. Functional analysis of bacterial communities provides insights into changes in microbially mediated benthic nutrient transformations associated with invasive dreissenid mussels and benthic algae in lake ecosystems.

The Genus Cladophora Kützing (Ulvophyceae) as a Globally Distributed Ecological Engineer.

The green algal genus Cladophora forms conspicuous nearshore populations in marine and freshwaters worldwide, commonly dominating peri-phyton communities. As the result of human activities, including the nutrient pollution of nearshore waters, Cladophora-dominated periphyton can form nuisance blooms. On the other hand, Cladophora has ecological functions that are beneficial, but less well appreciated. For example, Cladophora has previously been characterized as an ecological engineer because its complex structure fosters functional and taxonomic diversity of benthic microfauna. Here, we review classic and recent literature concerning taxonomy, cell biology, morphology, reproductive biology, and ecology of the genus Cladophora, to examine how this alga functions to modify habitats and influence littoral biogeochemistry. We review the evidence that Cladophora supports large, diverse populations of microalgal and bacterial epiphytes that influence the cycling of carbon and other key elements, and that the high production of cellulose and hydrocarbons by Cladophora-dominated periphyton has the potential for diverse technological applications, including wastewater remediation coupled to renewable biofuel production. We postulate that well-known aspects of Cladophora morphology, hydrodynamically stable and perennial holdfasts, distinctively branched architecture, unusually large cell and sporangial size and robust cell wall construction, are major factors contributing to the multiple roles of this organism as an ecological engineer.

Integrated photo-bioelectrochemical system for contaminants removal and bioenergy production.

An integrated photobioelectrochemical (IPB) system was developed by installing a microbial fuel cell (MFC) inside an algal bioreactor. This system achieves the simultaneous removal from a synthetic solution of organics (in the MFC) and nutrients (in the algal bioreactor), and the production of bioenergy in electricity and algal biomass through bioelectrochemical and microbiological processes. During the one-year operation, the IPB system removed more than 92% of chemical oxygen demand, 98% of ammonium nitrogen, and 82% of phosphate and produced a maximum power density of 2.2 W/m(3) and 128 mg/L of algal biomass. The algal growth provided dissolved oxygen to the cathode reaction of the MFC, whereas electrochemical oxygen reduction on the MFC cathode buffered the pH of the algal growth medium (which was also the catholyte). The system performance was affected by illumination and dissolved oxygen. Initial energy analysis showed that the IPB system could theoretically produce enough energy to cover its consumption; however, further improvement of electricity production is desired. An analysis of the attached and suspended microbes in the cathode revealed diverse bacterial taxa typical of aquatic and soil bacterial communities with functional roles in contaminant degradation and nutrient cycling.

The epiphytic microbiota of the globally widespread macroalga Cladophora glomerata (Chlorophyta, Cladophorales).

PREMISE OF THE STUDY: The filamentous chlorophyte Cladophora produces abundant nearshore populations in marine and freshwaters worldwide, often dominating periphyton communities and producing nuisance growths under eutrophic conditions. High surface area and environmental persistence foster such high functional and taxonomic diversity of epiphytic microfauna and microalgae that Cladophora has been labeled an ecological engineer. We tested the hypotheses that (1) Cladophora supports a structurally and functionally diverse epiphytic prokaryotic microbiota that influences materials cycling and (2) mutualistic host-microbe interactions occur. Because previous molecular sequencing-based analyses of the microbiota of C. glomerata found as western Lake Michigan beach drift had identified pathogenic associates such as Escherichia coli, we also asked if actively growing lentic C. glomerata harbors known pathogens. METHODS: We used 16S rRNA gene amplicon pyrosequencing to examine the microbiota of C. glomerata of Lake Mendota, Dane, Wisconsin, United States, during the growing season of 2011, at the genus- or species-level to infer functional phenotypes. We used correlative scanning electron and fluorescence microscopy to describe major prokaryotic morphotypes. KEY RESULTS: We found microscopic evidence for diverse bacterial morphotypes, and molecular evidence for ca. 100 distinct sequence types classifiable to genus at the 80% confidence level or species at the 96-97% level within nine bacterial phyla, but not E. coli or related human pathogens. CONCLUSIONS: We inferred that bacterial epiphytes of lentic C. glomerata have diverse functions in materials cycling, with traits that indicate the occurrence of mutualistic interactions with the algal host.

Rapid effects of diverse toxic water pollutants on chlorophyll a fluorescence: variable responses among freshwater microalgae.

Chlorophyll a fluorescence of microalgae is a compelling indicator of toxicity of dissolved water contaminants, because it is easily measured and responds rapidly. While different chl a fluorescence parameters have been examined, most studies have focused on single species and/or a narrow range of toxins. We assessed the utility of one chl a fluorescence parameter, the maximum quantum yield of PSII (F(v)/F(m)), for detecting effects of nine environmental pollutants from a range of toxin classes on 5 commonly found freshwater algal species, as well as the USEPA model species, Pseudokirchneriella subcapitata. F(v)/F(m) declined rapidly over <20 min in response to low concentrations of photosynthesis-specific herbicides Diuron(®) and metribuzin (both <40 nM), atrazine (<460 nM) and terbuthylazine (<400 nM). However, F(v)/F(m) also responded rapidly and in a dose-dependent way to toxins glyphosate (<90 µM), and KCN (<1 mM) which have modes of action not specific to photosynthesis. F(v)/F(m) was insensitive to 30-40 µM insecticides methyl parathion, carbofuran and malathion. Algal species varied in their sensitivity to toxins. No single species was the most sensitive to all nine toxins, but for six toxins to which algal F(v)/F(m) responded significantly, the model species P. subcapitata was less sensitive than other taxa. In terms of suppression of F(v)/F(m) within 80 min, patterns of concentration-dependence differed among toxins; most showed Michaelis-Menten saturation kinetics, with half-saturation constant (K(m)) values for the PSII inhibitors ranging from 0.14 µM for Diuron(®) to 6.6 µM for terbuthylazine, compared with a K(m) of 330 µM for KCN. Percent suppression of F(v)/F(m) by glyphosate increased exponentially with concentration. F(v)/F(m) provides a sensitive and easily-measured parameter for rapid and cost-effective detection of effects of many dissolved toxins. Field-portable fluorometers will facilitate field testing, however distinct responses between different species may complicate net F(v)/F(m) signal from a community.

Physiological responses of intertidal marine brown algae to nitrogen deprivation and resupply of nitrate and ammonium.

Intertidal macroalgae Fucus and Laminaria experience seasonally fluctuating inorganic N supply. This study examined the effects of long-term N deprivation, recovery following N resupply, and effects of elevated ammonium and nitrate exposure on N acquisition in intertidal algae using manipulations of N supply in tank culture. Over 15 weeks of N deprivation, internal N and nitrate reductase activity (NRA) declined, but maximum quantum yield of PSII was unaffected in Fucus serratus and Fucus vesiculosus. Low NRA was maintained despite no external nitrate availability and depletion of internal pools, suggesting a constitutive NRA, insensitive to N supply. Nitrate resupplied to N-starved thalli was rapidly taken up and internal nitrate pools and NRA increased. Exposure to elevated (50 microM) nitrate over 4 days stimulated nitrate uptake and NRA in Laminaria digitata and F. serratus. Exposure to elevated ammonium suppressed NRA in L. digitata but not in F. serratus. This novel insensitivity of NRA to ammonium in Fucus contrasts with regulation of NRA in other algae and higher plants. Ammonium suppression of NRA in L. digitata was not via inhibition of nitrate uptake and was independent of nitrate availability. L. digitata showed a higher capacity for internal nitrate storage when exposed to elevated ambient nitrate, but NRA was lower than in Fucus. All species maintained nitrate assimilation capacity in excess of nitrate uptake capacity. N uptake and storage strategies of these intertidal macroalgae are adaptive to life in fluctuating N supply, and distinct regulation of N metabolism in Fucus vs Laminaria may relate to position in the intertidal zone.

Nutrient limitation and morphological plasticity of the carnivorous pitcher plant Sarracenia purpurea in contrasting wetland environments.

* Plasticity of leaf nutrient content and morphology, and macronutrient limitation were examined in the northern pitcher plant, Sarracenia purpurea subsp. purpurea, in relation to soil nutrient availability in an open, neutral pH fen and a shady, acidic ombrotrophic bog, over 2 yr following reciprocal transplantation of S. purpurea between the wetlands. * In both wetlands, plants were limited by nitrogen (N) but not phosphorus (P) (N content < 2% DW(-1), N : P < 14) but photosynthetic quantum yields were high (F(V)/F(M) > 0.79). Despite carnivory, leaf N content correlated with dissolved N availability to plant roots (leaf N vs , r(2) = 0.344, P < 0.0001); carnivorous N acquisition did not apparently overcome N limitation. * Following transplantation, N content and leaf morphological traits changed in new leaves to become more similar to plants in the new environment, reflecting wetland nutrient availability. Changes in leaf morphology were faster when plants were transplanted from fen to bog than from bog to fen, possibly reflecting a more stressful environment in the bog. * Morphological plasticity observed in response to changes in nutrient supply to the roots in natural habitats complements previous observations of morphological changes with experimental nutrient addition to pitchers.

Seasonal variations in nitrate reductase activity and internal N pools in intertidal brown algae are correlated with ambient nitrate concentrations.

Nitrogen metabolism was examined in the intertidal seaweeds Fucus vesiculosus, Fucus serratus, Fucus spiralis and Laminaria digitata in a temperate Irish sea lough. Internal NO(3) (-) storage, total N content and nitrate reductase activity (NRA) were most affected by ambient NO(3) (-), with highest values in winter, when ambient NO(3) (-) was maximum, and declined with NO(3) (-) during summer. In all species, NRA was six times higher in winter than in summer, and was markedly higher in Fucus species (e.g. 256 +/- 33 nmol NO(3) (-) min(-1) g(-1) in F. vesiculosus versus 55 +/- 17 nmol NO(3) (-) min(-1) g(-1) in L. digitata). Temperature and light were less important factors for N metabolism, but influenced in situ photosynthesis and respiration rates. NO(3) (-) assimilating capacity (calculated from NRA) exceeded N demand (calculated from net photosynthesis rates and C : N ratios) by a factor of 0.7-50.0, yet seaweeds stored significant NO(3) (-) (up to 40-86 micromol g(-1)). C : N ratio also increased with height in the intertidal zone (lowest in L. digitata and highest in F. spiralis), indicating that tidal emersion also significantly constrained N metabolism. These results suggest that, in contrast to the tight relationship between N and C metabolism in many microalgae, N and C metabolism could be uncoupled in marine macroalgae, which might be an important adaptation to the intertidal environment.