Experiments were conducted in the mesopelagic subtropical northeast Atlantic Ocean to determine the range of variability in the prokaryotic leucine-to-carbon conversion factor (CF), and prokaryotic growth efficiency (PGE). The way prokaryotic heterotrophic production (PHP) is calcu- lated directly influences PGE (variations of PGE between 1 and 31% were found for a single sample). The empirically obtained deep-water CFs showed a 7-fold variability (0.13 to 0.85 kg C mol–1 Leu), but were always lower than the theoretical CF of 1.55 kg C mol–1 Leu assuming no isotope dilution. Empirically determined CFs were highly correlated to PGE, suggesting that both parameters are rep- resentations of the same basic metabolic processes. Overall, the PGEs obtained in this study suggest that mesopelagic prokaryotic assemblages can sometimes be as important in carbon processing as their epipelagic counterparts.
The distribution of prokaryotic abundance (PA), prokaryotic heterotrophic production (PHP), and suspended particulate organic material (POM), as well as total and dissolved (operationally defined as passing through 0.2 mu m pore size filters) potential extracellular enzymatic activities (EEA; alpha- and beta-glucosidase [AGase and BGase], leucine aminopeptidase [LAPase], and alkaline phosphatase [APase]) were determined in the meso- and bathypelagic waters of the (sub)tropical Atlantic along an eastern zonal transatlantic transect and a western N-S transect. Significant differences between both transects were found for POM concentration but not for PA, PHP (except in the subsurface and oxygen minimum layer), and dissolved and total EEA. PHP decreased by 3 orders of magnitude from the lower euphotic zone to bathypelagic waters, while PA and cell-specific PHP decreased only by 1 and 2 orders of magnitude, respectively. The proportion of the dissolved to the total EEA was high in the dark ocean for all the enzymes, ranging from 54 to 100, 56 to 100, 65 to 100 and 57 to 97 % for AGase, BGase, LAPase and APase, respectively. The kinetic parameters (V-max, and K-m) of both the dissolved and total fractions of LAPase and APase were very similar throughout the water column, suggesting a similar origin for both dissolved and particulate EEA. Significant correlations of both dissolved and total EEA were found with prokaryotic metabolism and the POM pool. Based on the previous notion that the fraction of dissolved EEA is higher in particle-attached than in free-living microbes, our results suggest that microbial activity in the dark ocean occurs mainly on colloidal and particulate material. This is in agreement with recent genomic evidence. However, these colloidal and particulate materials are prone to disruption during the sampling process. Hence, more selective sampling techniques are needed to specifically collect these deep-water aggregates that probably represent hotspots of microbial activity in the deep ocean.
The distribution of marine Crenarchaeota Group 1, marine Euryarchaeota Group II and some major groups of Bacteria (SAR 11, Roseobacter, Gammaproteobacteria and Bacteroidetes) was investigated in the North Atlantic water column (surface to 2000 m depth) along a transect from the coastal waters of the NW African upwelling to the offshore waters of the Canary Coastal Transition Zone (CTZ). Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) was used to describe the prokaryotic assemblages. Bulk picoplankton abundance and leucine incorporation were determined. Pronounced changes in prokaryotic assemblage composition were observed from the coast to the open ocean and at the deep chlorophyll maximum (DCM) with decreasing bulk heterotrophic activity. All bacterial groups decreased in absolute abundances from the coast to the open ocean; both archaeal groups increased towards the open ocean. Prokaryotic abundance and activity decreased 2 and 3 orders of magnitude, respectively, from the surface to 2000 m. Prokaryotic growth rates were high in the mesopelagic zone (similar to 0.13 d(-)1), compared to other reports from the central North Atlantic. SARI 1 in total picoplankton abundance decreased from 42 % in the DCM to 4 % at 2000 m, while marine Crenarchaeota Group I increased from 1 % in the DCM to 39 % in the oxygen minimum layer. A clear influence of the different intermediate water masses was observed on the bulk heterotrophic picoplankton activity, with lower leucine incorporation rates corresponding to layers where patches of Antarctic Intermediate Water were detected. Coast-ocean and surface-depth gradients in bulk prokaryotic abundance and production and assemblage composition were comparable to changes observed in basin-scale studies, pinpointing the CTZs as regions of strong variability in microbial diversity and metabolism.
The aim of this study was to determine the relative importance of the different processes/mechanisms by which the toxic haptophyte Prymnesium parvum, cultured under different nutrient conditions, affects non-toxic phytoplankton competitors and microzooplankton grazers. P. parvum was cultured under steady-state growth in different nutrient conditions: nitrogen depleted (-N), phosphorus depleted (-P) and balanced nitrogen and phosphorus (+NP). Cells from each nutrient condition and culture cell-free filtrates, alone and combined with non-toxic prey (Rhodomonas salina), were used as food for the rotifer Brachionus plicatilis. An additional experiment was carried out to test the effect of P. parvum cells and culture cell-free filtrate on R. salina. The highest haemolytic activity values were achieved by -P F parvum cultures, followed by -N. However, the negative effect of R parvum on R. salina and rotifers did not correlate with haemolytic activity but with the number of P. parvum cells. -N-cultured P. parvum were the most toxic for both R. salina and rotifers, followed by +NP. Therefore, haemolytic activity is not a good indicator of the total potential toxicity of R parvum. The growth rate of R. salina was negatively affected by cell-free filtrates but the effect of P, parvum predation was greater. Rotifers fed on both toxic and non-toxic algae, indicating that they did not select against the toxic alga. The P. parvum cell-free filtrate had an effect on B. plicatilis, although this was weak, B, plicatilis was also indirectly affected by P. parvum due to the negative effects of the toxic alga on their prey (R. salina). However, the greatest negative effect of P. parvum on the rotifers was due to ingestion of the toxic cells. Therefore, the phytoplankton competitor R. salina is more affected by P. parvum predation and the grazer B. plicatilis is more affected by ingestion of the toxic cells, the effects of excreted compounds being secondary.
Prymnesium parvum is a harmful algal bloom species present in many inland water bodies of the southcentral USA, but does not form fish-killing blooms in all of them. The present study tested the hypothesis that rotifer grazing of P. parvum might influence the incidence of blooms. Three-day in-lake experiments, which focused on the size fraction of zooplankton dominated by rotifers and natural phytoplankton assemblages inoculated with P. parvum, were conducted during the time of bloom development in 2 reservoirs of the southcentral USA: Lakes Somerville and Whitney, where the latter experiences P. parvum blooms and the former does not. Toxicity at a level lethal to fish was only occasionally observed during these experiments, so our experimental treatments are considered to be at a low-toxicity level. As a whole, rotifers in Lakes Somerville and Whitney selectively grazed P. parvum. Rotifers in Lake Somerville appeared to benefit from this selective grazing, while rotifers in Lake Whitney did not. The differences between rotifer communities from these lakes might be because rotifers from Lake Somerville historically have only been exposed to low levels of toxins produced by P. parvum and were able to develop resistance to these toxins, thus enabling them to persist and perhaps contribute to the suppression of blooms there. The opportunity for this type of microevolutionary adaptation may not occur in lakes where P. parvum blooms and waters reach high toxicity levels, such as those which have occurred historically in Lake Whitney.
Climate change is predicted to cause higher temperatures and increased precipitation, resulting in increased inflow of nutrients to coastal waters in northern Europe. This has been assumed to increase the overall heterotrophy, including enhanced bacterial growth. However, the relative importance of temperature, resource availability and bacterial community composition for the bacterial growth response is poorly understood. In the present study, we investigated effects of increased temperature on bacterial growth in waters supplemented with different nutrient concentrations and inoculated with microbial communities from distinct seasonal periods. Seven experiments were performed in the northern Baltic Sea spanning an entire annual cycle. In each experiment, bacterioplankton were exposed to 2 temperature regimes (in situ and in situ + 4 degrees C) and 5 nutrient concentrations. Generally, elevated temperature and higher nutrient levels caused an increase in the bacterial growth rate and a shortening of the response time (lag phase). However, at the lowest nutrient concentration, bacterial growth was low at all tested temperatures, implying a stronger dependence on resource availability than on temperature for bacterial growth. Furthermore, data indicated that different bacterial assemblages had varying temperature responses and that community composition was strongly affected by the combination of high nutrient addition and high temperature. These results support the concern that climate change will promote heterotrophy in aquatic systems, where nutrient levels will increase considerably. In such environments, the bacterial community composition will change, their growth rates will increase, and their response time will be shortened compared to the present situation.
Methionine (Met) and dimethylsulfoniopropionate (DMSP) are 2 important substrates that can serve as sources of sulfur and carbon to microbial communities in the sea. We studied the vertical and diel distributions and the assimilation rates of dissolved Met (dMet) and dissolved DMSP (dDMSP) into proteins of different microbial groups at Stn ALOHA, in the oligotrophic North Pacific Subtropical Gyre (NPSG). Concentrations of dMet never exceeded 50 pM and were at their daily minimum during the night-time (<0.17 pM). dMet assimilation into proteins accounted for <30% of the dMet lost from the dissolved pool, suggesting that other metabolic pathways were also important. Concentrations of dDMSP ranged from 0.35 to 1.0 nM in surface waters and did not present a distinguishable diel pattern. Cell-sorted Prochlorococcus, high nucleic acid (HNA), and low nucleic acid (LNA) non-pigmented bacteria showed a clear diel pattern for dMet and dDMSP assimilation, with higher rates during the night-time. Among the different groups, HNA bacteria had the highest per-cell assimilation rate for dMet and dDMSP, but when accounting for cell numbers in each group, the HNA and LNA bacterial group assimilation rates were comparable for both dDMSP and dMet. Integrated water column (0 to 125 m) dDMSP assimilation rates by the entire microbial assemblage were 1.7- To 5.3-fold faster than those for dMet, suggesting that dDMSP constitutes a more important source of sulfur than dMet to the microbial community of the NPSG during the time of our study.
Heterotrophic dinoflagellates bearing unicellular cyanobacterial symbionts are common within the order Dinophysiales. However, the ecological role of these symbionts is unclear. Due to the occurrence of such consortia in oceanic waters characterized by low nitrogen concentrations, we hypothesized that the symbionts fix gaseous nitrogen (N-2). Individual heterotrophic dinoflagellates containing cyanobacterial symbionts were isolated from the open Indian Ocean and off Western Australia, and characterized using light microscopy, transmission electron microscopy (TEM), and nitrogenase (nifH) gene amplification, cloning, and sequencing. Cyanobacteria, heterotrophic bacteria and eukaryotic algae were recognized as symbionts of the heterotrophic dinoflagellates. nifH gene sequences were obtained from 23 of 37 (62%) specimens of dinoflagellates (Ornithocercus spp. and Amphisolenia spp.). Interestingly, only 2 specimens contained cyanobacterial nifH sequences, while 21 specimens contained nifH genes related to heterotrophic bacteria. Of the 137 nifH sequences obtained 68% were most similar to Alpha-, Beta-, and Gammaproteobacteria, 8% clustered with anaerobic bacteria, and 5% were related to second alternative nitrogenases (anfH). Twelve sequences from 5 host cells formed a discrete cluster which may represent a not yet classified nifH cluster. Eight dinoflagellates contained only 1 type of nifH sequence (>99% sequence identity) but overall the putative N-2-fixing symbionts did not appear host specific and mixed assemblages were often found in single host cells. This study provides the first insights into the nifH diversity of dinoflagellate symbionts and suggests a symbiotic co-existence of non-diazotrophic cyanobacteria and N-2-fixing heterotrophic bacteria in heterotrophic dinoflagellates.
Cyanobacterial nitrogen-fixers (diazotrophs) play a key role in biogeochemical cycling of carbon and nitrogen in the ocean. In recent years, the unusual symbiotic diazotrophic cyanobacterium Atelocyanobacterium thalassa (UCYN-A) has been recognized as one of the major diazotrophs in the tropical and subtropical oceans. In this review, we summarize what is currently known about the geographic distribution of UCYN-A, as well as the environmental factors that govern its distribution. In addition, by compiling UCYN-A nifH sequences from the GenBank no. database as well as those from nifH gene amplicon next generation sequencing studies, we present an in-depth analysis of the distribution of defined UCYN-A sublineages (UCYN-A1, UCYN-A2 and UCYN-A3) and identify a novel sublineage, UCYN-A4, which may be significant in some environments. Each UCYN-A sublineage exhibited a remarkable global distribution pattern and several UCYN-A sublineages frequently co-occurred within the same sample, suggesting that if they represent different ecotypes they have overlapping niches. Recently, single cell visualization techniques using specific probes targeting UCYN-A1 and UCYN-A2 and their respective associated eukaryotic partner cells showed that the size of the consortia and the number of UCYN-A cells differed between these 2 sublineages. Combined, the results highlight that UCYN-A sublineages likely have different physiological requirements, which need to be accounted for in future studies. Furthermore, based on our increasing knowledge of the diversity of the UCYN-A lineage, we discuss some of the limitations of currently used cultivation-independent molecular techniques for the identification and quantification of UCYN-A.
We studied allelopathy in the dinoflagellate genus Alexandrium by testing the effect of A. tamarense on a natural plankton community from Hopavagen Bay, Trondheimsfjord, Norway, and the effect of toxic and non-toxic strains of A. tamarense and a toxic strain of A. minutum on algal monocultures. Also, a possible relation between the allelopathic effect and the production of paralytic shellfish poison (PSP) toxin was investigated. A. tamarense affected the whole phytoplankton community by decreasing the growth rate and changing the community structure (relative abundance of each species, dominant species). A negative effect of A. tamarense was also observed on ciliates, but not on bacteria numbers, In the bioassay with algal monocultures, the diatom Thalassiosira weissflogii and the cryptophyte Rhodomonas sp. were exposed to the filtrate of Alexandrium spp. All tested Alexandrium strains negatively affected T weissflogii and Rhodomonas sp. cultures, independent of whether PSP toxins were produced. The compounds released by Alexandrium caused lysis of natural and cultured algal cells, suggesting that the allelopathic effect may be connected with previously described ichthyotoxic and haemolytic properties of Alexandrium. Furthermore, the observation that several components of the plankton community were affected by compounds released by A. tamarense emphasizes the importance of allelopathy for the ecology of this species.
For aquatic systems, studies on allelopathic interactions among phytoplankton have increased over recent years, with the main focus on the role of the donor organism. In this study, we report on the response of a target organism to allelochemicals and whether this response was affected by stress conditions (nutrient limitation). We exposed the diatom Thalassiosira weissflogii, grown under different nitrogen (N) and phosphorus (P) conditions (NP, -N, or -P), to single or daily additions of a cell-free filtrate of Prymnesium parvum (grown with no nutrient limitation). When we exposed T weissflogii to a single addition of filtrate, all 3 treatments were inhibited by P. parvum. However, T weissflogii NP was the most resistant, while T weissflogii -N showed the highest sensitivity to P. parvum filtrate, followed by T weissflogii -P. When T weissflogii was exposed. to daily additions of P. parvum, the degree of inhibition of all T weissflogii treatments was higher than when only 1 initial addition was made. In this case, even the treatment that had the highest resistance (T weissflogii NP) was not only inhibited by the filtrate, but also showed a decrease in cell numbers. Nevertheless, T weissflogii -N was still more sensitive than the other treatments. Therefore, nutrient-limiting conditions may increase allelopathic effects, by making the target more susceptive to allelopathic compounds. Under these conditions, allelopathy may play a strong role in phytoplankton competition, especially in natural environments where the allelochemicals are continuously released and, thus, the target species do not have time to recover.
Domain-specific metabolic inhibitors are currently used to differentiate archaeal from bacterial activity. However, studies testing the specificity of these inhibitors are sparse or are based on cultured strains. We determined the inhibition specificity of erythromycin (EMY) and N1-guanyl-1,7-diaminoheptane (GC7) on bacterial and archaeal communities in the North Atlantic. EMY and GC7 are assumed to inhibit bacterial and archaeal activity, respectively. Heterotrophic prokaryotic activity was estimated via H-3-leucine incorporation on the cell-specific level using catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH). In the water masses studied, the contribution of Thaumarchaeota to total picoplankton abundance ranged from 5 to 24% while Euryarchaeota contributed 2 to 6%; the relative abundance of Bacteria ranged from 29 to 48%. The addition of EMY and GC7 reduced the bulk leucine incorporation by similar to 77% and similar to 41%, respectively. Evaluation of the inhibition efficiency of EMY on a cell-specific level showed no difference between Archaea (76.0 +/- 14.2% [SD]) and Bacteria (78.2 +/- 9.5%). Similarly, the reduction of substrate uptake in GC7-treated samples was similar in Archaea (59.9 +/- 24%) and Bacteria (47.2 +/- 9.6%). Taken together, our results suggest that in complex open-ocean prokaryotic communities neither EMY nor GC7 is efficient as a domain-specific inhibitor.
We now have a relatively good idea of how bulk microbial processes shape the cycling of organic matter and nutrients in the sea. The advent of the molecular biology era in microbial ecology has resulted in advanced knowledge about the diversity of marine microorganisms, suggesting that we might have reached a high level of understanding of carbon fluxes in the oceans. However, it is becoming increasingly clear that there are large gaps in the understanding of the role of bacteria in regulating carbon fluxes. These gaps may result from methodological as well as conceptual limitations. For example, should bacterial production be measured in the light? Can bacterial production conversion factors be predicted, and how are they affected by loss of tracers through respiration? Is it true that respiration is relatively constant compared to production? How can accurate measures of bacterial growth efficiency be obtained? In this paper, we discuss whether such questions could (or should) be addressed. Ongoing genome analyses are rapidly widening our understanding of possible metabolic pathways and cellular adaptations used by marine bacteria in their quest for resources and struggle for survival (e.g. utilization of light, acquisition of nutrients, predator avoidance, etc.). Further, analyses of the identity of bacteria using molecular markers (e.g. subgroups of Bacteria and Archaea) combined with activity tracers might bring knowledge to a higher level. Since bacterial growth (and thereby consumption of DOC and inorganic nutrients) is likely regulated differently in different bacteria, it will be critical to learn about the life strategies of the key bacterial species to achieve a comprehensive understanding of bacterial regulation of C fluxes. Finally, some processes known to occur in the microbial food web are hardly ever characterized and are not represented in current food web models. We discuss these issues and offer specific comments and advice for future research agendas.
Here, we examine the use of bacterial isolates growing in artificial media or seawater as a means to investigate bacterial activity in the upper ocean. The discovery of a major role of bacteria in the ocean's carbon cycle owes greatly to the development of culture-independent assemblage-level approaches; however, this should not detract from the recognition of model isolates as representing the environmental microbiome. A long-established tool for culturing bacteria, in medicine and general microbiology, has been agar plates. In addition, a great variety of liquid substrates including seawater have been used to successfully identify and cultivate important bacteria such as Pelagibacter ubique. Yet, the discrepancy between microscopic counts and plate counts, the great plate count anomaly, has led to a biased perception of the limited relevance of isolated bacteria. Linking isolates to whole-genome sequencing, phylogenetic analysis and computational modeling will result in culturable model bacteria from different habitats. Our main message is that bacterial ecophysiology, particularly growth rates in seawater, and functionalities inferred through the identity, abundance and expression of specific genes could be mechanistically linked if more work is done to isolate, culture and study bacteria in pure cultures. When we rally behind a strategy aimed at culturing targeted phenotypes, we are not saying that culture independent studies of bacteria in the sea are not informative. We are suggesting that culturebased studies can help integrate the ecological and genomic views.
Our understanding of microbial food web interactions in the ocean is essentially based on research performed in the euphotic layer, where the interactions between phytoplankton and prokaryotic plankton, mainly heterotrophic Bacteria, are well established. In the euphotic layer, particularly in meso- and eutrophic waters, prokaryotic plankton are mainly top-down controlled by bacterivorous flagellates and viruses, affecting metabolically active, fast growing populations more than dormant stages. In the meso- and bathypelagic realm of the ocean, however, prokaryotic plankton are thought to be mainly bottom-up controlled, because the heterotrophic component of the prokaryotic community is limited by the availability of organic carbon. However, deep-water prokaryotes exhibit a number of peculiarities compared to prokaryotes in the euphotic layer, among which are a large genome size and a gene repertoire indicative of a predominately surface-attached mode of life. This indicates that deep-water prokaryotic activity might be primarily associated with particles. Our present knowledge indicates that the microbial communities and their interactions in the deep ocean are likely very different from those known from surface waters. Increasing efforts to shed light on the microbial biota of the ocean's interior will likely lead to the discovery of novel metabolic pathways in prokaryotes and to the resolution of the current discrepancy between the geochemical evidence of remineralization rates of organic matter and actual measurements.
Increases in water colour (brownification) have been observed in aquatic systems in the Northern Hemisphere, partly caused by increased loading of organic carbon from terrestrial origins. We investigated the effect of increase in water colour on the composition, structure and function of lake plankton communities (bacteria, phytoplankton and zooplankton) conducting a mesocosm experiment in 3 medium-coloured lakes (average absorbance at 420 nm: 0.034 cm(-1)), with different nutrient concentrations and phytoplankton community composition. To simulate an increase in water colour, we added humic substances (HuminFeed) at 3 different concentrations. The additions significantly affected the water colour of the mesocosms, but had no measurable effect on total organic carbon concentration, thus change in light conditions was the main effect of our treatment on the plankton communities. The increase in water colour did not significantly affect the measured functions (productivity, respiration) and biomass of the plankton communities (bacteria, phytoplankton and zooplankton), but led to changes in the relative abundance of some phytoplankton taxa and, to a lesser extent, the bacterial community (differences in relative abundance). The treatments had no significant effect on zooplankton biomass or composition. Our study suggests that increases in water colour favour low-light-adapted phytoplankton species, which in turn also can affect bacterial composition, whereas the change in light climate had no clear impact on the functioning of plankton communities in weakly humic lakes.
Carbohydrate macromolecules (dextrans) labeled with fluorescein isothiocyanate (FITC) were taken up by the dinoflagellate Alexandrium catenella at a substrate concentration of 5 mg C l(-1) The FITC-labeled dextrans appeared in what resembled food vacuoles inside the dinoflagellate cells. Between 5 and 50% of the cells contained fluorescent green vacuoles. A. catenella took up dextrans of high molecular weight (2000 kDa) but did not show significant uptake of lower molecular weight (20 kDa) dextrans. The uptake of the 2000 kDa dextran was higher with addition of humic substances to the growth medium and did not seem related to the presence of bacteria. Phagocytosis of fluorescent microspheres (0.36 mu m) by A. catenella was also investigated. Although aggregation of fluorescent microspheres was observed in the sulcal region of the cells, no evidence was found of phagocytosis of bacterial-size prey by A. catenella. These observations show that A. catenella has the capacity to take up high molecular weight organic molecules. perhaps by pinocytosis.
Mixotrophy by the photosynthetic dinoflagellate Heterocapsa triquetra was investigated using fluorescently labelled algae (FLA) (size 1, 3, 6 mu m). Experiments were conducted in nitrogen-and phosphorus-replete/depleted medium under light and dark conditions. Incubations ranged from several hours to several days. The dinoflagellate was capable of phagotrophy when exposed to light and dark periods in nutrient-depleted medium. H. triquetra showed similar ingestion rates in the light (range: 0.1 to 0.4 FLA dino(-1) d(-1)) and in the dark (range: 0.06 to 0.4 FLA dino(-1) d(-1)). The dinoflagellate was able to selectively ingest the different FLA. The cyanobacterium Synechococcus sp, was not ingested, whereas a small round flagellate and the diatom Thalassiosira pseudonana were observed inside the cells. The flagellate was ingested at higher rates than the diatom in both the light and the dark. About 40% of the labelled flagellate was removed from the suspension by H. triquetra in the light and 22% in the dark. The diatom was removed from the suspension at the same rate (27 to 30%) in both light and dark treatments. After 5 d incubation in nutrient-depleted medium and no addition of FLA, the proportion of small H. triquetra cells (<1000 mu m(3)) increased from 50 to over 75%. In the presence of FLA, the distribution of H. triquetra cell volumes showed that the proportion of larger cells (>2000 mu m(3)) increased from 6 to >20% during long incubations in the light and the dark. Since the frequency of observed cells with ingested FLA varied from 3 to 20% for the same period, the proportion of larger cells may be the phagotrophic proportion of the population. However, only 8 (dark) to 12% (light) of the observed H. triquetra cell volume increase can be explained in terms of carbon from the ingestion of fluorescently labelled phytoplankton. We conclude that phagotrophy in H. triquetra may be important in maintaining the population in environments of low nutrient concentration and low light intensity.
We investigated whether or not the presence of copepods and different light conditions induced cyst formation in dinoflagellates. Scrippsiella trochoidea was exposed to Acartia tonsa directly and indirectly (grazer filtrate), in high light and low light conditions. The ingestion, faecal production and egg production of A. tonsa were compared between diets of S. trochoidea vegetative cells and temporary cysts. We found no effect of direct or indirect exposure to A. tonsa on S. trochoidea cyst formation in either high light or low light conditions. Controls and A. tonsa treatments kept in light displayed around 20% temporary cysts, whereas controls and A. tonsa treatments in low light were shown to have 50 to 80% temporary cysts. Thus, low light conditions had a strong effect on temporary cyst formation. No hypnocysts were observed in any experiment, which is probably related to the longer incubation times needed for their observation. Feeding on diets dominated by temporary cysts compared to vegetative cells decreased ingestion by a factor of 2.7, while faecal and egg production decreased by a factor of 2.2 and 2.9, respectively, suggesting that induction of temporary cysts in response to A. tonsa could be a survival strategy. However, S. trochoidea does not possess any grazer-induced defence in terms of temporary cyst formation, as it did not produce temporary cysts when exposed to A. tonsa. Rather, induction of temporary cysts seems to be controlled by decreased light intensity, which is a favorable trait for this species when driven to water depths where light is scarce.
We verified an active uptake of kleptoplastids in the toxic and bloom-forming dinoflagellatesof the genus Dinophysis from its preferred prey, the ciliate Myrionecta rubra, using a quantitativereal-time PCR technique. During a 65 d starvation/feeding experiment with Dinophysis caudata,changes in plastid 16S rRNA, plastid autofluorescence and plastid/nuclear DNA ratio throughthe cell cycle were followed with quantitative real-time PCR and flow cytometry. During starvation,the cultures initially showed a rapid growth and a 3.5-fold increase of number of cells ml–1, while atthe same time, plastid DNA cell–1 showed a 3.5-fold decrease, and a 3.6-fold decrease in phycoerythrinfluorescence cell–1. The decrease in plastid DNA cell–1 d–1 closely followed culture growth rate(Pearson correlation, r = 0.91), indicating that existing plastids were diluted within the growing populationand that no new plastids were synthesised by the cells. When starved cells were re-fed by theciliate M. rubra on Days 43 to 51 of the experiment, plastid DNA cell–1 increased 7-fold up to 14 00016S DNA copies per cell, thereby directly revealing the kleptoplastic behaviour. The implication isthat not only availability of the prey M. rubra itself, but also the supply of suitable kleptoplastidsmight be an important controlling factor for Dinophysis spp. bloom formation and decline.
Intracellular contents of carbon, nitrogen and phosphorus in phytoplankton cells are traditionally measured using concentrates containing thousands to millions of cells. In this study we have used a Nuclear MicroProbe (NMP) as an approach for the determination of C, N and P concentrations in single filaments of three cyanobacteria species: Anabaena sp., Nodularia spumigena and Aphanizomenon flos-aquae var. klebahnii isolated from Baltic Sea water. Estimations of C, N and P content per cell have been calculated and compared with the concentrations found with traditional bulk methods. No significant differences regarding the C, N and P cellular content were found between the two methods for each of the species tested (p < 0.05). From our results we conclude that the use of NMP can be a useful tool for studying the elemental contents in single phytoplankton cells
Dinophysis norvegica is a commonly occurring dinoflagellate species and a producer of diarrhetic shellfish poisons. Natural samples were collected from Trondheim fjord, Norway, to analyse nutrient (O, C, N, P) and toxin (dinophysitoxins [DXTs], okadaic acid [OA], pectenotoxins [PTXs]) content in D. norvegica cells. Nutrient and toxin analyses were also carried out on cells grown under nutrient-sufficient and nutrient-deficient conditions to determine how intracellular nutrient and toxin content varied under different nutrient availability conditions. Nutrient analyses were conducted using nuclear microprobe techniques that can accurately analyse single cells, and toxin analyses were carried out using liquid chromatography and mass spectroscopy. The intracellular carbon, nitrogen and phosphorus content in individual cells varied greatly, and intracellular C:N:P ratios showed that the cells were both N- and P-deficient when compared to the Redfield ratio. The ideal N:P ratio in the media for D. norvegica was found to be below the Redfield ratio, but intracellular ratios did not show a clear relationship with those in the media. N:P ratios of D. norvegica were higher than expected, which is likely due to their phagotrophy on zooplankon. The highest toxin values found were traces of PTX2, 24.72 pg PTX2SA cell(-1), 2.19 pg DTX1 cell(-1), and 1.01 pg OA cell(-1). However, we found no clear relationship between the content of intracellular nutrients and toxins.
Marine waters are generally considered to be nitrogen (N) limited and are therefore favourable environments for diazotrophs, i.e. organisms converting atmospheric N-2 into ammonium or nitrogen oxides available for growth. In some regions, this import of N supports up to half of the primary productivity. Diazotrophic Cyanobacteria appear to be the major contributors to marine N-2 fixation in surface waters, whereas the contribution of heterotrophic or chemoautotrophic diazotrophs to this process is usually regarded inconsequential. Culture-independent studies reveal that non-cyanobacterial diazotrophs are diverse, widely distributed, and actively expressing the nitrogenase gene in marine and estuarine environments. The detection of nifH genes and nifH transcripts, even in N-replete marine waters, suggests that N-2 fixation is an ecologically important process throughout the oceans. Because this process is highly sensitive to and inhibited by molecular oxygen (O-2), diazotrophy requires efficient scavenging of intracellular O-2 or growth in environments with low ambient O-2 concentration. Particles with interior low-O-2 micro-zones and oceanic oxygen minimum zones are just 2 potential habitats suitable for N-2 fixation by non-cyanobacterial diazotrophs. Our ignorance about the regulation of N-2 fixation by non-Cyanobacteria in their natural marine environments currently prevents an evaluation of their importance in marine N cycling and budgets. A review of the molecular data on distribution and expression of nifH genes in non-Cyanobacteria suggests that further study of the role of these Bacteria in N cycling at local, regional and global scales is needed.
Infection of marine dinoflagellates by the parasitic dinoflagellate Amoebophrya spp. plays an important role in population dynamics and carbon flow in marine food webs. It has been extensively reported that Amoebophrya parasitoids occur in temperate coastal areas of the northern hemisphere; however, little is known about their distribution and importance in tropical areas and southern oceans. We used an rRNA-based, fluorescent in situ hybridization assay to detect Amoebophrya spp. infections during the decline of a late-summer dinoflagellate population dominated by Ceratium falcatiforme in a tropical coastal area of the southern Atlantic Ocean subjected to recurrent upwelling-downwelling cycles. Conditions during our survey were typical of downwelling when oligotrophic waters dominate the area. C. falcatiforme was the most infected host, with a prevalence averaging 2% over the study area at the beginning of sampling. At a fixed sampling station monitored over 4 wk, Amoebophrya prevalence escalated from 1 to 7% over a 6 d period, concomitant to a 94% decrease in host cell numbers. Infection by Amoebophrya was estimated to have killed ca. 11% of the host cell population within this period; thus, parasitism was not the main factor behind the C. falcatiforme population decline. Estimates based on biovolume calculations indicate that ca. 6.5% of the carbon in the decaying C. falcatiforme population was transformed into parasitoid dinospores, which became available to tintinnid ciliates that were abundant during our survey. Such a trophic link might be relevant in tropical oligotrophic waters when the system is characterized by a microbial food web structure.
Although temperature is a key parameter controlling the activity and growth of all microorganisms, information about how water temperature may structure the bacterioplankton community is not consistent. We examined the relationship between temperature and the community composition, cell volume, and morphology of marine bacterioplankton in 4 continuous cultures harbouring multispecies communities. All 4 cultures were maintained at a turnover time of 0.04 h(-1) but at different temperatures of 10, 15, 20, and 25 degrees C. Denaturing gradient gel electrophoresis analyses showed that the community composition shifted in response to temperature. Cell volumes were determined from digital photomicrographs using an image analysis program, which also allowed the identification of 3 morphological types of bacteria: cocci-, rod-, and vibrio-shaped bacteria. Mean bacterial cell volume decreased with increasing temperature, e.g., by 39% when the temperature was increased from 10 degrees C to 20 degrees C. When the temperature increased, the bacterial morphology also shifted from dominance by rod- and vibrio-shaped bacteria to dominance by coccoid bacteria. The results clearly indicate the potential role of temperature in driving the community succession of bacterioplankton and in selecting for smaller cells at higher temperatures.
To obtain insights into the coupling between community composition, diversity and community function, bacterioplankton assemblages from the Gulf of Trieste (Northern Adriatic Sea) were exposed to increasing environmental stress throughout 2 wk in continuous seawater cultures to construct communities differing in composition and diversity. The assemblages were exposed to (1) decreased temperature, (2) decreased temperature and phosphate addition or (3) decreased temperature, phosphate addition and lowered oxygen level. Bacterial and viral abundances as well as bacterial community composition stabilized during the second week of the experiment. Denaturing gradient gel electrophoresis and pyrosequencing of 16S rRNA genes showed dramatic reductions in bacterial diversity in all treatments and major compositional differences relative to the inoculum. Nevertheless, no differences in the ability to exploit dissolved organic carbon (DOC) were found for the acquired communities relative to the inoculum, indicating that the bacterial communities were functionally redundant. We speculate that oscillations in exploitation of the DOC pool in situ are mainly governed by factors limiting the overall bacterial growth, rather than perturbations affecting only subsets of the microbial biota.
The haptophyte PrymnesiuM parvum Carter is toxic and frequently responsible for harmful algal blooms in coastal waters. It is a mixotrophic species having the capability to feed on various planktonic microorganisms. It is frequently suggested that mixotrophic algae may obtain inorganic nutrients through phagotrophy and that nutrient depletion should then lead to increased food uptake. To study this, we investigated the feeding activity of P. parvum in semi-continuous, nutrient-limited cultures, using the cryptophyte Rhodomonas baltica as prey. P. parvum showed to be an active predator under all conditions investigated. After 2 h of incubation with prey, 40% of P. parvum cells were either feeding or contained recently formed food vacuoles. However, under the conditions used, no difference in feeding activity was found between treatments. On the contrary, the feeding activity was similar in P. parvum cultures that had been grown under N-limiting, P-limiting, N- and P-limiting, as well as under nutrient-replete conditions. It cannot be excluded that P. parvum under limiting nutrient conditions may acquire nutrients to be used in photosynthetic growth through phagotrophy. It is evident, however, that the species also feeds when inorganic nutrients are present in concentrations sufficient to support maximum phototrophic growth.
Nodularia spumigena Mertens ex Bornet & Flahault 1886 (Cyanophyceae) frequently forms harmful blooms in the Baltic Sea, and the toxin nodularin has been found in calanoid copepods during the blooms. Although nodularin has been found at higher trophic levels of the food web, no available information exists about the role of the microbial loop in the transfer of nodularin. We followed the transfer of nodularin to the copepod Eurytemora affinis during conditions that resembled initial 'pre-bloom' (Expt 1) and late stationary (Expt 2) phases of a N. spumigena bloom. The experiments were carried out using natural plankton communities spiked with cultured N. spumigena and grown in laboratory mesocosms, and E. affinis, which were isolated from the Baltic Sea and had no prior contact with nodularin. The plankton community was divided into 6 size fractions as follows: <150, <45, <20, <10, <3 and <0.2 pm, in which E. affinis was incubated for 24 h. Ingestion and clearance rates, food selection and faecal pellet production were based on microscopical analyses. Nodularin was measured with HPLC-MS with electrospray ionization in the copepods, as well as in dissolved and particulate fractions before and after incubation. We found that nodularin accumulated in copepods in all the plankton size fractions. The copepods contained nodularin concentrations of 14.3 +/- 11.6 (mean +/- SD) and 6.6 +/- 0.7 pg ind.(-1) after incubation in the < 150 mu m fraction in Expt 1 and Expt 2, respectively, while the range in the smaller size fractions was from 1.3 +/- 2.8 to 5.7 +/- 1.3 pg ind.-1. Nodularin was transferred to the copepods through 3 pathways: (1) by grazing on filaments of small N. spumigena, (2) directly from the dissolved pool, and (3) through the microbial food web by copepods grazing on ciliates, dinoflagellates and heterotrophic nanoflagellates. The relative importance of direct grazing on small N. spumigena filaments varied from moderate to insignificant. The microbial loop was important in nodularin transfer to higher trophic levels. Our results suggest that the importance of the microbial loop in harmful algal bloom (HAB) toxin transfer may be underestimated both in marine and freshwater systems.
To test the hypothesis that dissolved organic nitrogen (DON) is important for sustaining microphytobenthic (MPB) primary production during nitrogen-limited conditions, the uptake of 15N-labelled urea, the amino acids glycine (GLY) and glutamic acid (GLU), and nitrate and ammonium were measured under simulated in-situ light and temperature conditions. Microphytobenthic primary production and chlorophyll a (Chl a) were also measured. MPB was dominated by diatoms attached to sand grains, cyanobacteria making up ~30% of the biomass. Activities of the hydrolytic ectoenzymes leucin aminopeptidase (AMA), alkaline phosphatase (APA), and β-glucosidase (GLA) in filter-fractionated sediment showed that the microbenthic community was strongly N deficient, with the bacterial fraction (<1 µm) also phosphorus limited. DON uptake (urea + glutamic acid + glycine) accounted for ~ 50–65% of the uptake of 15N-labeled substrates, with higher proportion of DON uptake at low substrate concentrations (≤2 µM). Except for nitrate, the kinetics fitted a linear model. Calculated relative preference index (RPI) based on porewater concentrations, suggested that the order of preference of the microbenthic community was NH4+ > urea > GLU > NO3- > GLY. Using a prokaryotic inhibitor (chloramphenicol) and theoretical calculations of algal uptake based on C/Chl a ratios, it was estimated that “algal” nitrogen uptake accounted for ~ 55-90% of DON uptake. Uptake rates were, however, estimated to cover only 26% –50% of the N-demand of MPB, suggesting that porewater N concentrations were not sufficient to meet the microalgal demand in early summer and that MPB in sandy sediments of micro-tidal waters may often be severely N-limited.
The episodic hyperproduction of mucilage macroaggregates in the northern Adriatic Sea creates an important site for the accumulation, transformation, and degradation of organic matter. In this review, the structure and function of macroaggregate components in relation to their macrogel and colloidal fractions are discussed. High resolution electron microscopy showed a very complex structure, a honeycomb-like structure of the mucus macroagregates that might grow to macroscopic sizes. The process of the formation and microbial interaction with the physicochemical diversity of the organic matter pool is poorly understood. Whether the in situ bacteria react to the carbohydrate-rich mucus as an imbalance in its C:N:P ratio or whether the mucus is in fact largely a bacterial construct in relation to high dissolved organic carbon levels is unknown. The majority of carbohydrate and protein macroaggregate pools are potentially degradable, while the great majority of lipids can be preserved in the water column and exported away or finally deposited on the seabed. Our present knowledge indicates that different macroaggregate fractions and components are subjected to compositional selective reactivity, with important implications for macroaggregate persistence. Future work should reconcile the discrepancies between bacterial ectoenzyme potential activities and biogeochemical degradation sequences based on actual measurements. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies, has profound implications for the function of these complex communities. We need to improve our understanding of the dynamic relationship among bacteria, other microorganisms, and a variety of organic matter forms.
Ostreopsis ovata is a benthic dinoflagellate that produces palytoxin and its analogues.Since the end of the 1990s, toxic blooms of O. ovata have been recorded in many tropicaland temperate marine waters. These blooms often kill benthic invertebrates and cause healthproblems for humans. We hypothesize that increases in seawater temperature might induce theseblooms. A strain of O. ovata isolated from the southern coast of Japan was selected for study. O.ovata cells were exposed to 7 different temperatures from 24 to 30°C for 30 d, and growth rateswere noted. The specific growth rate was found to be highest at 25°C, next highest at 24°C andlower at 26, 28, 27, 30 and 29°C, in that order. The hypothesis that increased seawater temperaturecauses increases in growth rate was thus not supported. The cell toxicity and car bohydrate productionof O. ovata were highest at the temperature range that is optimal for cell growth. Increasesingsea surface temperature, as a result of global warming, is therefore not likely to have asubstantial effect on the bloom formation and toxicity of this Japanese strain of O. ovata.
The present study investigates the effect of brackish (7 PSU) and marine (26 PSU) salinity on physiological parameters and intra- and extracellular toxicity in 4 strains of Prymnesium parvum Carter. The different P. parvum strains were grown in batch cultures in 2 trials under different experimental conditions to test the development of intra- and extracellular toxicity during growth. The response of P. parvum toxicity to salinity was validated using 2 protocols. Intra-specific variations in growth rate, maximal cell density (yield) and cell morphology were controlled by salinity. Extracellular toxicity was higher at 7 PSU in all strains, but no correlation was found between intra- and extracellular toxicity. The variation of extracellular toxicity in response to salinity was much greater than that of intracellular toxicity, which indicates that P. parvum may be producing a variety of substances contributing to its various types of 'toxicity'.