Alternative nutritional strategies in protists: symposium introduction and a review of freshwater protists that combine photosynthesis and heterotrophy

The alternative nutritional strategies in protists that were addressed during the symposium by that name at the 2010 annual meeting of the International Society of Protistologists and here in contributed papers, include a range of mechanisms that combine photosynthesis with heterotrophy in a single organism. Often called mixotrophy, these multiple trophic level combinations occur across a broad range of organisms and environments. Consequently, there is great variability in the physiological abilities and relative importance of phototrophy vs. phagotrophy and/or osmotrophy in mixotrophic protists. Recently, research papers addressing ecological questions about mixotrophy in marine systems have been more numerous than those that deal with freshwater systems, a trend that is probably partly due to a realization that many harmful algal blooms in coastal marine systems involve mixotrophic protists. After an introduction to the symposium presentations, recent studies of mixotrophy in freshwater systems are reviewed to encourage continuing research on their importance to inland waters.     

Contrasting Mixotrophic Lifestyles Reveal Different Ecological Niches in Two Closely Related Marine Protists

Many marine microbial eukaryotes combine photosynthetic with phagotrophic nutrition, but incomplete understanding of such mixotrophic protists, their functional diversity, and underlying physiological mechanisms limits the assessment and modeling of their roles in present and future ocean ecosystems. We developed an experimental system to study responses of mixotrophic protists to availability of living prey and light, and used it to characterize contrasting physiological strategies in two stramenopiles in the genus Ochromonas. We show that oceanic isolate CCMP1393 is an obligate mixotroph, requiring both light and prey as complementary resources. Interdependence of photosynthesis and heterotrophy in CCMP1393 comprises a significant role of mitochondrial respiration in photosynthetic electron transport. In contrast, coastal isolate CCMP2951 is a facultative mixotroph that can substitute photosynthesis by phagotrophy and hence grow purely heterotrophically in darkness. In contrast to CCMP1393, CCMP2951 also exhibits a marked photoprotection response that integrates non-photochemical quenching and mitochondrial respiration as electron sink for photosynthetically produced reducing equivalents. Facultative mixotrophs similar to CCMP2951 might be well adapted to variable environments, while obligate mixotrophs similar to CCMP1393 appear capable of resource efficient growth in oligotrophic ocean environments. Thus, the responses of these phylogenetically close protists to the availability of different resources reveals niche differentiation that influences impacts in food webs and leads to opposing carbon cycle roles.     

Distributions and Biomass of Benthic Ciliates,Foraminifera and Amoeboid Protists in Marine,Brackish, and Freshwater Sediments

The quantitative importance of ciliates, foraminifers, and amoebae was investigated in marine, brackish, and freshwater sediments from 15 littoral stations. Total protozoan communities were usually dominated by ciliates in term of abundance, while amoebae often dominated in terms of biomass. Applying the biomass‐metabolic rate equation, ciliates, amoebae, and foraminifera were estimated to contribute 66% of the total abundance and 33% of the biomass, but up to 55% of the combined metabolic rate to the micro‐ and meiobenthos in the 15 sediments. Statistical analyses using ciliate data demonstrated: (1) species composition and community structures represented significant differences between freshwater and marine/brackish sediments, and subsequently between temperate and arctic sampling sites; (2) the occurrence of dominant ciliates and their allocation to feeding types indicated that herbivory was the most common feeding strategy in these sediments; (3) multivariate analyses showed all of the tested environmental factors (temperature, salinity, silt/clay, carbon, nitrogen, and chlorophyll a) to be important to varying degrees, but especially the combination of salinity, temperature, and silt/clay. Multiple factor effects or comprehensive influences might be important in regulating the distribution of protozoa in sediments. The importance of protozoa in sediment systems and the potential ecological significance of cysts are discussed.     

Protists with different feeding modes change biofilm morphology

The effect of Dexiostoma (filter feeder), Vannella, Chilodonella (raptorial feeders), Spumella , and Neobodo (direct interception feeders) on the morphology of multispecies bacterial biofilms was investigated in small flow cells. The filter feeder Dexiostoma campylum did not alter biofilm volume and porosity but stimulated the formation of larger microcolonies compared with ungrazed biofilms. In contrast, the raptorial feeder Vannella sp. efficiently grazed bacteria from the biofilm surface, leading to smaller microcolonies and lower maximal and basal layer thickness compared with ungrazed biofilms. Microcolony formation was not stimulated in the presence of the sessile Spumella sp. Chilodonella uncinata rasped bacteria from the outer surface leading to mushroom-shaped microcolonies. In the presence of C. uncinata and Spumella sp., the biofilm volume was 2.5–6.3 times lower compared with ungrazed biofilms. However, the biofilm porosity and the ratio of biofilm surface area to biofilm volume were 1.5–3.7 and 1.2–1.8 times higher, respectively. Thus, exchange of nutrients and gases between the biofilm and its surrounding fluid should also be improved in deeper biofilm layers, hence accelerating microbial growth.     

Mixotrophic cryptophytes and their predators in the Dry Valley lakes of Antarctica

1. The ingestion rates of planktonic, mixotrophic cryptophytes in two perennially ice-covered Antarctic lakes in the McMurdo Dry Valleys, were investigated during the summer of 1997–1998.
2. In Lake Fryxell, which is meromictic, ingestion rates increased with depth in November and were highest in a cryptophyte maximum close to the chemocline. In Lake Hoare, which is unstratified and freshwater, there was no significant difference in ingestion rates with depth. In both lakes, the highest ingestion rates occurred in early summer, decreasing in December and January. Ingestion rates varied between 0.2 bacteria cell⁻¹ h⁻¹ and 3.6 bacteria cell⁻¹ h⁻¹.
3. During November, mixotrophic cryptophytes removed up to 13% of bacterial biomass day⁻¹ and had a greater grazing impact than heterotrophic nanoflagellates (HNAN). As summer progressed, the grazing impact of cryptophytes and HNAN became similar.
4. The maximum depth of cryptophytes in Lake Fryxell was predated by a population of the ciliate Plagiocampa. Plagiocampa had an ingestion rate of 0.13–0.19 cryptophytes cell⁻¹ h⁻¹. The grazing impact on the cryptophyte community was insignificant. However, the ciliate appeared to be indulging in temporary mixotrophy, sequestering the cryptophytes for a number of weeks before digesting them.
5. It is suggested that mixotrophy is an important survival strategy in the extreme lake ecosystems of the McMurdo Dry Valleys.     

Mixotrophic ciliates in an Andean lake: dependence on light and prey of an Ophrydium naumanni population

1. Planktonic ciliates were examined during a spring–summer period (November 1998–April 1999) in the ultraoligotrophic Lake Moreno Oeste (41°5' S and 71°33' W, 758 m a.s.l), which belongs to the Nahuel Huapi System (Patagonia, Argentina). The lake is deep ( Z _max=90 m) and warm monomictic.
2. Sampling was performed at a mid-lake station, where vertical profiles of temperature and light were measured in situ , and samples for bacteria and ciliates enumeration were taken throughout the water column.
3. The peritrich Ophrydium naumanni , a freshwater pelagic ciliate with endosymbiotic Chlorella , was the dominant ciliate in the lake.
4.  Ophrydium naumanni and autotrophic picoplankton exhibit a clear coincidence in their vertical distribution ( P  < 0.05), preferring levels at or near the 1% of surface photosynthetically active radiation (PAR) irradiance. Both may have the same light requirements, or the coincidence may reflect a trophic relationship.
5. Dependence on light and prey by O. naumanni were studied using field experiments, in which we analysed ciliate grazing on bacteria, and in laboratory experiments, in which we compared particle uptake under dark and light conditions.
6.  Ophrydium naumanni was able to ingest particles [latex microspheres and fluorescently labelled bacteria (FLB)] in field and laboratory experiment, indicating that it has the potential to affect bacteria population of Lake Moreno Oeste.
7. Ciliate particle ingestion was observed to be dependent on light availability because under dark conditions, the ingestion was lowered ( P  < 0.05).     

Functional Ecology of Two Contrasting Freshwater Ciliated Protists in Relation to Temperature

We conducted microcosm experiments with two contrasting freshwater ciliates on functional traits (FTs) related to their growth rate (numerical response, NR) and ingestion rate (functional response, FR) over a range of ecologically relevant temperatures. Histiobalantium bodamicum and Vorticella natans are common planktonic ciliates but their abundance, swimming behavior, and temperature tolerance are different. In contrast to most sessile peritrich species, the motile V. natans is not strictly bacterivorous but also voraciously feeds upon small algae. We observed three main alterations in the shape of NR of both species with temperature, that is, change in the maximum growth rate, in the initial slope and in the threshold food level needed to sustain the population. Similarly, maximum ingestion rate, gross growth efficiency (GGE), and cell size varied with temperature and species. These findings caution against generalizing ciliate performance in relation to the ongoing global warming. Our results suggest that V. natans is the superior competitor to H. bodamicum in terms of temperature tolerance and bottom-up control. However, the abundance of V. natans is usually low compared to H. bodamicum and other common freshwater ciliates, suggesting that V. natans is more strongly top-down controlled via predation than H. bodamicum. The taxonomic position of V. natans has been debated. Therefore, to confirm species and genus affiliation of our study objects, we sequenced their small subunit ribosomal RNA (SSU rDNA) gene.     

The significance of inter- and intraspecific variation in bacterivorous and herbivorous protists

This paper reviews the emerging evidence on the significance of inter- and intraspecific variation in the feeding behaviour of aquatic protists. Small heterotrophic nanoflagellates (HNF) have been identified as the primary bacterial consumers in most aquatic environments. Recent research using novel techniques such as flow cytometry and high resolution video microscopy revealed that their feeding strategies and grazing rates are diverse. There is an important conceptual difference between uptake rates measured in short-term (min to h) experiments and grazing rates averaged over a longer-term (d). This is because the latter are strongly affected by digestion rates which are species-specific, i.e. the same bacterial prey may be digested differently by various grazers, and the same predator may selectively digest variable prey. Planktonic ciliates are the most important algal consumers in many lakes and marine systems. Large species-specific differences in their feeding behaviour and growth rates have been documented for closely related species. Intraspecific variation, which is, most likely, caused by varying clonal composition may be as important as interspecific variation. Finally, there is some evidence that the individual variability within a given population is generally large, both among bacterivorous HNF and among herbivorous ciliates. The consequences of this diversity becoming apparent at the levels of the species, population, clone and individual need to be considered by aquatic ecologists in their conceptual models. This revised version was published online in August 2006 with corrections to the Cover Date.     

Costs, benefits and characteristics of mixotrophy in marine oligotrichs

1. Oligotrich ciliates are an important part of most marine plankton communities. Mixotrophic (chloroplast-sequestering) oligotrichs, a common component of marine oligotrich communities, obtain fixed carbon from both photosynthesis as well as the ingestion of particulate food. Mixotrophy, in general, is often considered an adaptation permitting exploitation of food-poor environments. We examined the hypothesis that, among oligotrichs, mixotrophs may be at a disadvantage relative to heterotrophs in food-rich conditions in a nutrient-enrichment experiment. We compared growth responses of mixotrophic and heterotrophic oligotrichs in natural communities from the N.W. Mediterranean Sea in microcosms with daily nutrient additions resulting in increases in nanoflagellates and Synechococcus populations. The results indicated that both mixotrophic and heterotrophic oligotrichs respond to prey increases with rapid growth (μ=1.2 d−1).
2. To examine the hypothesis that the proportion of mixotrophic to heterotrophic oligotrichs changes with the trophic status of a system, increasing with oligotrophy, we examined data from a variety of marine systems. Across systems ranging in chlorophyll concentration from about 0.1 to 40 μg L−1, oligotrich cell concentrations are correlated with chlorophyll concentrations, and mixotrophs are a consistent component of oligotrich communities, averaging about 30% of oligotrich cell numbers.
3. We discuss the costs, benefits and possible uses of mixotrophy in marine oligotrichs and suggest that mixotrophy in marine oligotrichs is not closely linked to the exploitation of food-poor environments, but probably serves a variety of purposes.     

A Fossilized Microcenosis In Triassic Amber

ABSTRACT Detailed data on bacterial and protistan microfossils are presented from a 0.003 mm³ piece of Triassic amber (Schlierseerit, Upper Triassic period, 220-230 million years old). This microcenosis, which actually existed as such within a very small, probably semiaquatic habitat, included the remains of about two bacteria species, four fungi ( Palaeodikaryomyces baueri, Pithomyces -like conidia, capillitium-like hyphae, yeast cells) two euglenoids, two chlamydomonas ( Chlamydomonas sp., Chloromonas sp.), two coccal green microalgae ( Chlorella sp., Choricystis -like cells), one zooflagellate, three testate amoebae ( Centropyxis aculeata var. oblonga-like, Cyclopyxis eurystoma -like, Hyalosphenia baueri n. sp.), seven ciliates ( Pseudoplaryophrya nana -like, Mykophagophrys terricola -like, Cyrtolophosis mucicola -like, Paracondylostoma sp., Bryometopus triquetrus -like. Tetrahymena rostrata -like, Paramecium triassicum n. sp.) the microfossils correspond to or diverge from extant species only slightly.     

A Symbiont-Produced Protein and Bacterial Symbiosis in Amoeba proteus

ABSTRACT. Gram- symbiotic X-bacteria present in the xD strain of Amoeba proteus as required cell components, synthesize and export a large amount of a 29-kDa protein (S29x) into the host's cytoplasm across bacterial and symbiosome membranes. The S29x protein produced by E. coli transformed with the s29x gene is also rapidly secreted into the culture medium. Inside amoebae, S29x enters the host's nucleus as detected by confocal and irnmunoelectron microscopy, although it is not clear if S29x is selectively accumulated inside the nucleus. The deduced amino-acid sequence of S29x has a stretch of basic amino acids that could act as a nuclear localization signal, but there is no signal peptide at the N-terminus and the transport of S29x is energy independent. The functions of S29x are not known, but in view of its prominent presence inside the amoeba's nucleus, S29x is suspected to be involved in affecting the expression of amoeba's nuclear gene(s).     

Abundance and Community Structure of Picoplankton and Protists in the Microbial Food Web of Barguzin Bay,Lake Baikal

We examined the vertical abundance of bacteria, phytoplankton and protists along a transect of six stations from near-shore (Stn. 1) to off-shore (Stn. 6) in Barguzin Bay of Lake Baikal, in the summer of 2002. Chlorophyll concentrations at Stn. 1 were higher (>10μg l⁻¹) than at the other five stations (<3μg l⁻¹). Planktonic and sessile diatoms dominated at Stn. 1, while pico-phytoplankon was dominant at other stations. Densities of heterotrophic bacteria were high in both the epilimnion and the thermocline at all stations. Nanoflagellates were abundant in the epilimnion, and ciliates in the thermocline, but no horizontal trend could be found for these heterotrophs. At Stn. 1, not only filter feeding (Strombidium and Strobilidium) and raptorial (Balanion) ciliates but also predatory ciliates (Prorodon and Spathidiosus) dominated, while at other stations only the filter feeding and raptorial ciliates were dominant. In off-shore stations (Stns. 5 and 6), significant correlations were detected between concentrations of chlorophyll a and density of filter feeding or raptorial ciliates, suggesting tight food linkages between phytoplankton and these ciliates. The concentration of dissolved organic carbon (DOC) was significantly correlated with chlorophyll a concentration, although there was no significant correlation between DOC concentration and bacterial density. We suggest that there is a shift of the dominant food linkage from a herbivorous food chain in near-shore areas to a microbial food web in off-shore areas in Barguzin Bay of Lake Baikal.     

Bacteria and protozoa as integral components of the forest ecosystem--their role in creating a naturally varied soil fertility

The paper explores interactions between the two first organism groups to appear on earth, the bacteria and protozoa, and their interplay with the rest of the ecosystem focusing upon northern boreal forests. The microbial loop is suggested as a mechanism for local inputs of new N to the ecosystem. The possibility to couple short-term microbial processes with their long-term effects, — as registered in plants, soil and the atmosphere, via the abiotic variables — is explored. The latter are investigated in relation to the environments they create for the micro-organisms, and how this results in varying soil fertility. A chain of events is presented that relate high Ca concentration in the mineral soil and high water availability to increased nitrogen availability for plants via the micro-organisms. An example is given of the influence of these parameters directly upon protozoa along an extreme fertility gradient, and also indirect evidence from a Finnish field study of 30 sites with four fertility levels. Finally, there is a discussion about ways to convert knowledge gained in detailed studies of microbial interactions into forms useful when evaluating the present status of and effects of ameliorative management on ecosystems strongly affected by humans. This revised version was published online in August 2006 with corrections to the Cover Date.     

Spatio-temporal Investigations on the Planktonic Organisms of the Middle Loire (France), During the Low Water Period: Biodiversity and Community Dynamics

The objective of this work was to analyse the distribution of the planktonic communities involved in the functioning of a 255-km river stretch and to get a better understanding of the influence of the river morphology on the diversity and dynamics of the micro-organisms. The planktonic communities (phytoplankton, bacterioplankton, proto- and metazooplankton) scarcely considered together in fluvial systems, were analysed at four sites of the Middle Loire during the low water period, in parallel to physical and chemical analyses. Physical and chemical variables such as turbidity, pH, suspended matter and chlorophyll a concentration were high, illustrating the classical, productive summer period. The algae played a major role in the water oxygenation until end-summer, then the algal drop concomitant to the bacterial sustained abundance appeared responsible for oxygen depletion. The downstream site enriched by nutrients inputs of two tributaries, carried the highest algal and bacterial densities. Situated in a meanders zone, the Middle Loire is characterised by a high habitat heterogeneity, the up- and downstream sites were wide and spread of vast standing zones and vegetated islands, whereas the two intermediate ones were narrower and more uniform. This morphological variability strongly impacted the micro-organisms diversity and distribution. Indeed, the algae and zooplankton composition were clearly influenced by the physical habitats of the river, the Cyanophyta were favoured by the lentic conditions and the Bacillariophyta by the turbulent ones, while the young stage of copepod and the large rotifer predators were indicator of a lentic origin. Thereafter, the river heterogeneity interfered with the zooplankton dynamics, the standing conditions enhancing the rotifer predation. In that way, we hypothesise that two opposite patterns characterised the wide sites spread of lentic water and the more uniform channels. In the first case, the zooplankton could prey on the ciliates protozoan, which in return favoured the flagellate ones; conversely in the second situation the zooplankton limited by the physical constraints did not impact the ciliates which could depress the flagellates. Thus, the similar geomorphology of the distant upstream and downstream sites (255-km apart) induced relatively close organisms distribution. Hence, disagreeing with the river continuum concept, this assertion shows the strong influence played by the local morphological characteristics of the Middle Loire in potamoplankton composition and dynamics.     

Feeding by the Newly Described Mixotrophic Dinoflagellate Paragymnodinium shiwhaense: Feeding Mechanism,Prey Species,and Effect of Prey Concentration

ABSTRACT. To investigate the feeding by the newly described mixotrophic dinoflagellate Paragymnodinium shiwhaense (GenBank accession number=AM408889), we explored the feeding process and the kinds of prey species that P. shiwhaense is able to feed on using several different types of microscopes, including a transmission electron microscope and high‐resolution video‐microscopy. In addition, we measured the growth and ingestion rates of P. shiwhaense on its optimal algal prey Amphidinium carterae as a function of prey concentration. We also measured these parameters for edible prey at a single concentration at which the growth and ingestion rates of P. shiwhaense on A. carterae were saturated. Paragymnodinium shiwhaense feed on algal prey using a peduncle after anchoring the prey by a tow filament. Among the algal prey offered, P. shiwhaense ingested small algal species that had equivalent spherical diameters (ESDs) ≤11 μm (e.g. the prymnesiophyte Isochrysis galbana, the cryptophytes Teleaulax sp. and Rhodomonas salina, the raphidophyte Heterosigma akashiwo, and the dinoflagellates Heterocapsa rotundata and A. carterae). However, it did not feed on larger algal species that had ESDs ≥12 μm (e.g. the dinoflagellates Prorocentrum minimum, Heterocapsa triquetra, Scrippsiella trochoidea, Alexandrium tamarense, Prorocentrum micans, Gymnodinium catenatum, Akashiwo sanguinea, and Lingulodinium polyedrum) or the small diatom Skeletonema costatum. The specific growth rates for P. shiwhaense feeding upon A. carterae increased rapidly with increasing mean prey concentration before saturating at concentrations of ca. 350 ng C/ml (5,000 cells/ml). The maximum specific growth rate (i.e. mixotrophic growth) of P. shiwhaense on A. carterae was 1.097/d at 20 °C under a 14:10 h light–dark cycle of 20 μE/m²/s, while its growth rate (i.e. phototrophic growth) under the same light conditions without added prey was ?0.224/d. The maximum ingestion and clearance rates of P. shiwhaense on A. carterae were 0.38 ng C/grazer/d (5.4 cells/grazer/d) and 0.7 μl/grazer/h, respectively. The calculated grazing coefficients for P. shiwhaense on co‐occurring Amphidinium spp. was up to 0.07/h (i.e. 6.7% of the population of Amphidinium spp. was removed by P. shiwhaense populations in 1 h). The results of the present study suggest that P. shiwhaense can have a considerable grazing impact on algal populations.     

Responses of Very Shallow Marine Ecosystems to Nutrient Enrichment

The success of simple predictive relationships such as the Vollenweider plot in limnology has encouraged marine ecologists to attempt to develop similar models relating pollutant inputs to ecological conditions in estuaries. Most of these efforts have focused on relatively deep (>5?m) river mouth estuaries and embayments where primary production is dominated by phytoplankton. Experimental nutrient enrichment studies of phytoplankton-based mesocosms at the Marine Ecosystems Research Laboratory (MERL) have confirmed that simple Vollenweider type relationships can be found between the rate of input of inorganic nutrients and annual mean chlorophyll concentrations and primary production. However, much of the coastline of the U.S. is characterized by estuarine ecosystems that are very shallow, and where most of the primary production is carried out by angiosperms, such as eelgrass, Zostera marina, epiphytic algae, drift and attached macroalgae, and epibenthic microalgae, rather than by phytoplankton. We have not been able to find useful relationships between nutrient input and the type of plant providing most of the primary production or between nutrient input and the amount of primary production in such shallow lagoon systems. Attempting to adjust nutrient loading for varying hydraulic residence time did not improve the models. Experimental studies using shallow lagoon mesocosms have shown that there is a large variation in the abundance of the various plant forms in these very shallow systems, and that simple Vollenweider models are not likely to emerge for this type of environment. However, it does seem that total system production increases with nutrient enrichment at very low rates of input, and that eelgrass does not persist when exposed to even moderate levels of fertilization. Zostera responds to inorganic nitrogen enrichment and to shading by increasing the rate of leaf elongation and decreasing the allocation of resources to below ground roots and rhizomes. This reduces or eliminates lateral branching of the rhizomes and causes a decline in the density of shoots. Based on mesocosm studies, we propose several indicators of eelgrass health, including the rate of leaf elongation, plant density, and the shoot: root biomass ratio that all deserve further study and field testing.     

Pacific Salmon,Nutrients, and the Dynamics of Freshwater and Riparian Ecosystems

Pacific salmon (Oncorhynchus spp.) accumulate substantial nutrients in their bodies as they grow to adulthood at sea. These nutrients are carried to predominantly oligotrophic lakes and streams, where they are released during and after spawning. Research over more than 3 decades has shown that the annual deposition of salmon-borne marine-derived nutrients (MD-nutrients) is important for the productivity of freshwater communities throughout the Pacific coastal region. However, the pathways and mechanisms for MD-nutrient transfer and accumulation in freshwater and riparian ecosystems remain virtually unexplored, consequently, there are many uncertainties in this area. This article addresses three related topics. First, we summarize recent advances in our understanding of the linkages among MD-nutrients, freshwater (including riparian) ecosystems, and community dynamics by addressing the importance of MD-nutrients to lakes and streams and by then reviewing large-scale and long-term processes in the atmosphere and ocean that govern variability in salmon populations. Second, we evaluate the validity of the discoveries and their implications for active ecosystem management, noting areas where extrapolation from these results still requires great caution. Finally, we outline five key research issues where additional discoveries could greatly augment our understanding of the processes shaping the structure and dynamics of salmon populations and the characteristics of their freshwater habitat and associated riparian zones. Collectively, the data suggest that the freshwater portion of the salmon production system is intimately linked to the ocean. Moreover, for the system to be sustainable, a holistic approach to management will be required. This holistic approach will need to treat climate cycles, salmon, riparian vegetation, predators, and MD-nutrient flowpaths and feedbacks as an integrated system. Received 3 July 2001; accepted 14 December 2001.