Fluorescently Labeled Bacteria Research Articles

A human lectin array for characterizing host-pathogen interactions

A human lectin array has been developed to probe the interactions of innate immune receptors with pathogenic and commensal micro-organisms. Following the successful introduction of a lectin array containing all of the cow C-type carbohydrate-recognition domains (CRDs), a human array described here contains the C-type CRDs as well as CRDs from other classes of sugar-binding receptors, including galectins, siglecs, R-type CRDs, ficolins, intelectins and chitinase-like lectins. The array is constructed with CRDs modified with single-site biotin tags, ensuring that the sugar-binding sites in CRDs are displayed on a streptavidin-coated surface in a defined orientation and are accessible to the surfaces of microbes. A common approach used for expression and display of CRDs from all of the different structural categories of glycan-binding receptors allows comparisons across lectin families. In addition to previously documented protocols for binding of fluorescently-labeled bacteria, methods have been developed for detecting unlabeled bacteria bound to the array by counter-staining with DNA-binding dye. Screening has also been undertaken with viral glycoproteins and bacterial and fungal polysaccharides. The array provides an unbiased screen for sugar ligands that interact with receptors and many show binding not anticipated from earlier studies. For example, some of the galectins bind with high affinity to bacterial glycans that lack lactose or N-acetyllactosamine. The results demonstrate the utility of the human lectin array for providing a unique overview of the interactions of multiple classes of glycan-binding proteins in the innate immune system with different types of micro-organisms.

A novel in-vivo phagocytosis assay to gain cellular insights on sponge-microbe interactions

IntroductionSponges harbor diverse, specific, and stable microbial communities, but at the same time, they efficiently feed on microbes from the surrounding water column. This filter-feeding lifestyle poses the need to distinguish between three categories of bacteria: food to digest, symbionts to incorporate, and pathogens to eliminate. How sponges discriminate between these categories is still largely unknown. Phagocytosis is conceivable as the cellular mechanism taking part in such discrimination, but experimental evidence is missing. We developed a quantitative in-vivo phagocytosis assay using an emerging experimental model, the sponge Halichondria panicea.MethodsWe incubated whole sponge individuals with different particles, recovered the sponge (host) cells, and tracked the incorporation of these particles into the sponge cells. Fluorescence-activated cell sorting (FACS) and fluorescent microscopy were used to quantify and verify phagocytic activity, defined here as the population of sponge cells with incorporated particles. Sponges were incubated with a green microalgae to test if particle concentration in the seawater affects the percentage of phagocytic activity, and to determine the timing where the maximum of phagocytic cells are captured in a pulse-chase experiment. Lastly, we investigated the application of our phagocytic assay with other particle types (i.e., fluorescently-labelled bacteria and fluorescent beads).Results and discussionThe percentage of sponge cells that had incorporated algae, bacteria, and beads ranged between 5 to 24%. These phagocytic sponge cells exhibited different morphologies and sizes depending on the type of particle presented to the sponge. Particle incorporation into sponge cells was positively related to algal concentration in the seawater, suggesting that sponge cells adjust their phagocytic activity depending on the number of particles they encounter. Our results further revealed that sponge phagocytosis initiates within minutes after exposure to the particles. Fluorescent and TEM microscopy rectified algal internalization and potential digestion in sponge cells. To our knowledge, this is the first quantitative in-vivo phagocytosis assay established in sponges that could be used to further explore phagocytosis as a cellular mechanism for sponges to differentiate between different microorganisms.

Effects of equine SALSA on neutrophil phagocytosis and macrophage cytokine production.

Salivary scavenger and agglutinin (SALSA) is a secreted protein with various immunomodulatory roles. In humans, the protein agglutinates and inactivates microorganisms, and inhibits the release of pro-inflammatory cytokines. Saliva, which is rich in SALSA, accelerates bacterial phagocytosis, but SALSA’s contribution is unclear. In horses, the functions of SALSA in inflammation remain undetermined, so they were investigated through phagocytosis and cytokine assays. Equine SALSA was purified from duodenal tissue, which contains abundant SALSA. To assess phagocytosis, fluorescently-labelled bacteria were incubated with 20, 10, 5, or 2.5 μg/mL of SALSA or phosphate buffered saline (PBS), and then incubated at 37°C or on ice with whole blood from seven healthy horses. Fluorescence was measured by gating on neutrophils using a flow cytometer, and compared between groups. To assess effects on cytokine production, alveolar macrophages were isolated from bronchoalveolar lavage fluid of five healthy horses and cultured in serum-free media for 24 hours with different concentrations of SALSA plus 1 μg/mL lipopolysaccharide (LPS), only LPS, or only media. Cytokines were measured in supernatant using an equine-specific multiplex bead immunoassay. There was significantly greater phagocytosis in samples incubated at 37°C compared to incubation on ice. Samples incubated with 20 μg/mL of SALSA at 37°C had less phagocytosis compared to samples with 10 or 2.5 μg/mL SALSA, or PBS. Alveolar macrophages incubated with SALSA plus LPS released significantly less CXC motif chemokine ligand 1, interleukin-8, interleukin-10, and tumor necrosis factor α, and more granulocyte colony stimulating factor (G-CSF), compared to macrophages incubated with LPS alone. These findings indicate anti-inflammatory effects, which may be due to interference with toll-like receptor 4 recognition of LPS or downstream signaling. Increase in G-CSF following incubation with SALSA suggests a novel mechanism for immunoregulation of alveolar macrophages by SALSA, addressing a knowledge gap regarding its functions in horses.

Comparison of two fluorescent probes in preclinical non-invasive imaging and image-guided debridement surgery of Staphylococcal biofilm implant infections

Implant-associated infections are challenging to diagnose and treat. Fluorescent probes have been heralded as a technologic advancement that can improve our ability to non-invasively identify infecting organisms, as well as guide the inexact procedure of surgical debridement. This study’s purpose was to compare two fluorescent probes for their ability to localize Staphylococcus aureus biofilm infections on spinal implants utilizing noninvasive optical imaging, then assessing the broader applicability of the more successful probe in other infection animal models. This was followed by real-time, fluorescence image-guided surgery to facilitate debridement of infected tissue. The two probe candidates, a labelled antibiotic that targets peptidoglycan (Vanco-800CW), and the other, a labelled antibody targeting the immunodominant Staphylococcal antigen A (1D9-680), were injected into mice with spine implant infections. Mice were then imaged noninvasively with near infrared fluorescent imaging at wavelengths corresponding to the two probe candidates. Both probes localized to the infection, with the 1D9-680 probe showing greater fidelity over time. The 1D9-680 probe was then tested in mouse models of shoulder implant and allograft infection, demonstrating its broader applicability. Finally, an image-guided surgery system which superimposes fluorescent signals over analog, real-time, tissue images was employed to facilitate debridement of fluorescent-labelled bacteria.

Bacterial Adhesion Kinetics in a High Throughput Setting in Seconds-minutes Time Resolution.

Bacterial surface adhesion, the first step in many important processes including biofilm formation and tissue invasion, is a fast process that occurs on a time scale of seconds. Adhesion patterns tend to be stochastic and spatially heterogeneous, especially when bacteria are present in low population densities and at early stages of adhesion to the surface. Thus, in order to observe this process, a high degree of temporal resolution is needed across a large surface area in a way that allows several replicates to be monitored. Some of the current methods used to measure bacterial adhesion include microscopy, staining-based microtiter assays, spectroscopy, and PCR. Each of these methods has advantages in assaying aspects of bacterial surface adhesion, but none can capture all features of the process. In the protocol presented here, adapted from Shteindel et al., 2019 , fluorescently-labeled bacteria are monitored in a multi-titer setting using a standard plate fluorimeter and a dye that absorbs light in the fluorophore excitation and emission wavelengths. The advantage of using this dye is that it restricts the depth of the optic layer to the few microns adjacent to the bottom of the microtiter well, eliminating fluorescence originating from unattached bacteria. Another advantage of this method is that this setting does not require any preparatory steps, which enables reading of the sample to be repeated or continuous. The use of a standard multi-titer well allows easy manipulation and provides flexibility in experimental design.

Detection of Phagotrophy in the Marine Phytoplankton Group of the Coccolithophores (Calcihaptophycidae, Haptophyta) During Nutrient-replete and Phosphate-limited Growth.

Mixotrophic algae that combine photoautotrophy with phagotrophy in a single cell are prevalent in marine ecosystems. Here, we assessed the ability of food ingestion in coccolithophores, an important group of calcifying haptophytes inhabiting the oceans. We tested four species from different coccolithophore lineages (Emiliania huxleyi, Calcidiscus leptoporus, Coccolithus braarudii, and Calyptrosphaera sphaeroidea). For both E.huxleyi and C.leptoporus we included different life phases (haploid and diploid). For C.braarudii we only tested diploid heterococcolithophore cells, while for C.sphaeroidea we only tested haploid holococcolithophore cells. Phagotrophy was assessed using fluorescently labeled bacteria (FLB) as model prey item, under nutrient-replete and phosphate-limited conditions. We detected by microscopy ingestion of FLB by all species, except the diploid C.braarudii strain. However, a previous study detected ingestion by haploid cells of C.braarudii. These overall results indicate that mixotrophy and the ability to ingest prey is widespread in coccolithophores. Yet, in all tested species the ingestion of FLB was low (<1% of the population contained prey at all time points over 2days), namely for E.huxleyi and the diploid cells from C.leptoporus where detection of ingestion was sporadic. Moreover, no clear differences were detected between life phases in E.huxleyi and C.leptoporus under equal circumstances, or between replete and limited growth conditions.

A Novel Lactic Acid Bacteria Mixture: Macrophage-Targeted Prophylactic Intervention in Colorectal Cancer Management.

Colorectal cancer (CRC) is one of the most common forms of cancer. Its onset from chronic inflammation is widely accepted. Moreover, dysbiosis plays an undeniable role, thus the use of probiotics in CRC has been suggested. They exhibit both anti- and pro-inflammatory properties and restore balance in the microbiota. The aim of this study was to investigate the immunomodulatory properties of six lactobacilli with probiotic features in an in vitro model of macrophage-like cells and to test these pooled probiotics for their anti-tumour properties in a chemically induced CRC model using Wistar male rats. Upon co-culture of M1- and M2-like macrophages with lactobacilli, cytokine release (TNF-α, IL-1β, IL-18, IL-23) and phagocytic activity using fluorescent-labelled bacteria were tested. The effects of orally administered probiotics on basic cancer and immune parameters and cytokine concentration (TNF-α, IL-1β, IL-18) in colon tumours were studied. Tested lactobacilli exhibited both pro- and anti-inflammatory properties in in vitro conditions. In vivo study showed that the administration of probiotics was able to decrease multiplicity, volume and total tumour numbers, restore colon length (p < 0.05) and increase IL-18 production (p < 0.05) in tumour tissue. These data indicate both an immunomodulatory effect of probiotics on distinct macrophage subsets and a protective effect against chemically-induced CRC.

Importance of mixotrophic flagellates during the ice-free season in lakes located along an elevational gradient

Mixotrophy seems to be widespread among phytoplankton, but whether this strategy is more relevant in oligotrophic lakes remains unclear. Here, we tested the hypothesis that the relative abundance of mixotrophic flagellates in lakes increases along an elevational gradient paralleling increasingly oligotrophic conditions. For this purpose, 12 lakes located between 575 and 2796 m above sea level were sampled in summer and fall to include two different seasonal windows in phytoplankton dynamics and environmental conditions. The degree of mixotrophy in phytoplankton was estimated in tracer experiments using fluorescently-labeled bacteria and done with composite samples collected in the euphotic zone and in samples obtained from the chlorophyll-a maximum. The results indicated the existence of a positive trend particularly in summer in the relative abundance of mixotrophic flagellates with elevation, however, this trend was not linear, and exceptions along the elevational gradient were found. Changes in the relative abundance of mixotrophic flagellates were related with significant changes in water transparency, DOC and phosphorus concentrations, as well as in bacterial and flagellate abundance. Overall, our results reveal that the harsh growth conditions found in oligotrophic high mountain lakes favor a mixotrophic trophic strategy among phytoplankton.

Abiotic and Biotic Factors Affecting the Ingestion Rates of Mixotrophic Nanoflagellates (Haptophyta)

Mixotrophic haptophytes comprise one of several important groups of mixotrophic nanoflagellates in the pelagic environment. This study aimed to investigate if phagotrophy in mixotrophic haptophytes is regulated by light or other factors in the surface (SE) and bottom (BE) of the euphotic zone in the subtropical northwestern Pacific Ocean. We estimated the rates of bacterial ingestion by haptophytes using fluorescently labeled bacteria (FLBs) and fluorescence in situ hybridization. Haptophyte diversity and abundance were also investigated in the same sampling area. The annual mean abundance of haptophytes was 419 ± 85.6cellsmL-1 in both SE and BE. Cells 3-5μm in size were the dominant group in all haptophytes and accounted for majority of bacteria standing stock removed by haptophytes (53%). Most haptophyte ingestion rates (IRs) were not significantly different between the two layers (average SE ingestion rate: 12.5 ± 2.29bacHap-1h-1; BE: 14.7 ± 3.03bacHap-1h-1). Furthermore, the haptophyte IRs were negatively correlated with nitrate concentrations in the SE and positively correlated with bacterial abundances in the BE, which accounts for the significantly high IRs in August 2012 and 2013. These findings imply that mixotrophic haptophytes in this region had different factors affecting phagotrophy to adapt to the ambient light intensity alterations between SE and BE.

CLIQ-BID: A method to quantify bacteria-induced damage to eukaryotic cells by automated live-imaging of bright nuclei

Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements. Here, we describe a simple and cost-effective method based on automated analysis of live cells with stained nuclei, which allows real-time quantification of bacteria-induced eukaryotic cell damage at single-cell resolution. We demonstrate that this automated high-throughput microscopy approach permits screening of libraries composed of interference-RNA, bacterial strains, antibodies and chemical compounds in ex vivo infection settings. The use of fluorescently-labelled bacteria enables the concomitant detection of changes in bacterial growth. Using this method named CLIQ-BID (Cell Live Imaging Quantification of Bacteria Induced Damage), we were able to distinguish the virulence profiles of different pathogenic bacterial species and clinical strains.

"In vitro" evaluation of the bacterivore potential of three Cladoceran species occurring in tropical and subtropical regions.

Cladocerans occupy an important position among the bacterial consumers in freshwater food webs, being relatively non-selective filter feeders that can exert a strong impact on microbial populations. We evaluated the bacterivory potential of three cladocerans species of subtropical and tropical occurrence (Ceriodaphnia silvestrii, Daphnia gessneri and Ceriodaphnia cornuta) at different life stages, by scanning electron microscopy (SEM) and by the quantification of their bacterial consumption rates using fluorescently labeled bacteria (FLB). All species showed filter mesh sizes sufficiently narrow to retain bacteria. The largest distance between setules was found in primiparous C. cornuta, and the smallest in neonate D. gessneri. The lowest ingestion (IR) and removal (RR) rates were observed in primiparous C. silvestrii and the highest values were observed in adults of D. gessneri. We conclude that bacteria may be considered an important food supplement, particularly for neonates who presented smaller intersetular distances and higher filtration rates than the other life stages.

Distribution of mixotrophic nanoflagellates along the latitudinal transect of the central North Pacific

To elucidate the ecological importance of mixotrophic nanoflagellates in the open ocean and the environmental factors that regulate their abundance, we surveyed latitudinal distributions of autotrophic, mixotrophic and heterotrophic nanoflagellates in the central North Pacific Ocean along a transect at 170°W. Mixotrophic nanoflagellates significantly contributed (26–64 %) to total bacterivory, as measured by the fluorescently-labeled bacteria method, from the equatorial through the subarctic regions, which reinforces the importance of mixotrophic nanoflagellates as a trophic link in the open ocean. The proportion of mixotrophic to total plastidic nanoflagellates was significantly higher in the nutrient-depleted subtropical gyre than in other regions, sometimes exceeding 10 %. Additionally, the proportion was negatively correlated with soluble reactive phosphorus concentration within the tropical and subtropical waters, suggesting that low nutrient availability could facilitate phagotrophy of plastidic nanoflagellates, which may explain the survival of nano-sized eukaryotic phytoplankton in the ultraoligotrophic water. In the subarctic regions, the proportion exhibited no obvious relationship with any environmental parameter. Conversely, the numerical proportion of mixotrophic nanoflagellates in total phagotrophic nanoflagellates (sum of mixotrophic and heterotrophic nanoflagellates) was positively correlated with nutrient concentrations. In contrast to macronutrient availability, the physical stability of the water column did not appear to affect the contribution of mixotrophic nanoflagellates.

Eicosanoids up-regulate production of reactive oxygen species by NADPH-dependent oxidase in Spodoptera exigua phagocytic hemocytes

Eicosanoids mediate cellular immune responses in insects, including phagocytosis of invading microbes. Phagocytosis entails two major steps, the internalization of microbes and the subsequent killing of them via formation of reactive oxygen species (ROS). Here, we posed the hypothesis that eicosanoids mediate ROS production by activating NADPH-dependent oxidase (NOX) and tested the idea in the model insect, Spodoptera exigua. A NOX gene (we named SeNOX4) was identified and cloned, yielding a full open reading frame encoding 547 amino acid residues with a predicted molecular weight of 63,410Da and an isoelectric point at 9.28. A transmembrane domain and a large intracellular domain containing NADPH and FAD-binding sites were predicted. Phylogenetic analysis indicated SeNOX4 clusters with other NOX4 genes. SeNOX4 was expressed in all life stages except eggs, and exclusively in hemocytes. Bacterial challenge and, separately, arachidonic acid (AA, a precursor of eicosanoid biosynthesis) injection increased its expression. The internalization step was assessed by counting hemocytes engulfing fluorescence-labeled bacteria. The phagocytic behavior was inhibited by dsRNA suppression of SeNOX4 expression and, separately by dexamethasone (DEX, a specific inhibitor of eicosanoid biosynthesis) treatments. However, injecting AA to dsSeNOX4-treated larvae did not rescue the phagocytic activity. Hemocytic ROS production increased following bacterial challenge, which was sharply reduced in dsSeNOX4-treated, and separately, in DEX-treated larvae. AA partially reversed the suppressed ROS production in dsSeNOX4-treated larvae. Treating larvae with either the ROS-suppressing dsSeNOX4 construct or DEX rendered experimental larvae unable to inhibit bacterial proliferation in their hemocoels. We infer that eicosanoids mediate ROS production during phagocytosis by inducing expression of SeNOX4.

The importance of protozoan bacterivory in a subtropical environment (Lobo-Broa Reservoir, SP, Brazil).

This study evaluated the importance of heterotrophic nanoflagellates (HNF) and ciliates bacterivory in a mesotrophic subtropical environment (Lobo-Broa Reservoir, Brazil) by the quantification of their ingestion rates. The in situ experiments using fluorescently labelled bacteria (FLB) were carried out bimonthly over one year (three surveys in the dry season and three in the rainy one) at the sub-surface of two sampling points that have different trophic degrees. The ingestion rates for both ciliates and HNF were higher in the meso-eutrophic region (point 2) due to the higher water temperatures, which accelerate the metabolism of protozoans and the higher bacteria densities. Concerning total protozoan bacterivory, the HNF had the greatest grazing impact on bacterial community, especially the HNF <5 µm. The data showed that HNF grazing, in addition to regulating the bacteria abundance, also induced changes to the bacterial community structure, such as increasing size and numbers of bacterial filaments. The ciliates were also important to the system bacterivory, especially in point 2, where there were high densities and ingestion rates. The protozoan bottom-up control was more important in the dry season and the top-down control was more important in the rainy season, so, these two forces are equally important to the bacterial abundance regulation in this reservoir in an annual basis.

Ingestion of bacteria in a eutrophic subtropical reservoir pond with food web mainly controlled by zooplankton grazing

This study evaluated the zooplanktonic bacterivory at a eutrophic subtropical reservoir pond by the quantification of the bacterial grazing and clearance rates of the protozooplanktonic (ciliates and nanoplankton) and metazooplanktonic (rotifers, cladocerans and copepods) populations during one year period. For this purpose, in situ experiments with fluorescently labeled bacteria (FLB) were carried out every two months on the sub-surface of the reservoir pond. Considering the individual grazing and clearance rates, the metazooplanktonic organisms showed the highest consumption of bacteria. However, in terms of population and considering all the zooplanktonic community, the heterotrophic nanoplanktonic organisms (HNP) accounted for 73% of the total bacteria ingested, being the most important bacterial consumers in the reservoir, due to their high population densities. Among them, the HNP smaller than 5 μm showed the highest population grazing rates, also due to their high abundance. These organisms were the main responsible for bacteria regulation by grazing in the reservoir. Among the metazooplanktonic organisms, the highest ingestion of bacteria occurred by the copepods (10%) during the wet season, and by the rotifers (22%) during the dry season. Thus, the metazooplanktonic population grazing rates were significantly different over the year, between the cold/dry and hot/rainy season. These seasonal differences were not observed in the density and biomass of picoplankton nor in the population grazing rates of ciliates and HNP. Nevertheless, the protozoa (ciliates and HNP) were directly responsible for most of the predation on bacteria, while the metazooplanktonic populations were indirectly responsible for it by the consumption of protozoa in a cascading effect.

Molecular Cloning and Functional Characterization of the Dual Oxidase (BmDuox) Gene from the Silkworm Bombyx mori

Reactive oxygen species (ROS) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and their related dual oxidases are known to have significant roles in innate immunity and cell proliferation. In this study, the 5,545 bp cDNA of the silkworm Bombyx mori dual oxidase (BmDuox) gene containing a full-length open reading frame was cloned. It was shown to include an N-terminal signal peptide consisting of 28 amino acid residues, a 240 bp 5′-terminal untranslated region (5′-UTR), an 802 bp 3′-terminal region (3′-UTR), which contains nine ATTTA motifs, and a 4,503 bp open reading frame encoding a polypeptide of 1,500 amino acid residues. Structural analysis indicated that BmDuox contains a typical peroxidase domain at the N-terminus followed by a calcium-binding domain, a ferric-reducing domain, six transmembrane regions and binding domains for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD). Transcriptional analysis revealed that BmDuox mRNA was expressed more highly in the head, testis and trachea compared to the midgut, hemocyte, Malpighian tube, ovary, fat bodies and silk glands. BmDuox mRNA was expressed during all the developmental stages of the silkworm. Subcellular localization revealed that BmDoux was present mainly in the periphery of the cells. Some cytoplasmic staining was detected, with rare signals in the nucleus. Expression of BmDuox was induced significantly in the larval midgut upon challenge by Escherichia coli and Bombyx mori nucleopolyhedrovirus (BmNPV). BmDuox-deleted larvae showed a marked increase in microbial proliferation in the midgut after ingestion of fluorescence-labeled bacteria compared to the control. We conclude that reducing BmDuox expression greatly increased the bacterial load, suggesting BmDuox has an important role in inhibiting microbial proliferation and the maintenance of homeostasis in the silkworm midgut.

Relative importance of phototrophic, heterotrophic, and mixotrophic nanoflagellates in the microbial food web of a river-influenced coastal upwelling area

The contribution of phototrophic nanoflagellates (PNF) as primary producers and het- erotrophic (HNF) and mixotrophic (MNF) nanoflagellates as major grazers of bacterioplankton was assessed during a 3 yr study in a highly productive, river-influenced coastal upwelling area under contrasting seasons (winter/non-upwelling vs. spring/upwelling). Sampling was conducted at 2 stations — around a river plume and at an intensive seasonal upwelling site — with contrasting en- vironmental gradients. The MNF were evaluated functionally for the possession of photo- pigments and experimentally for the ability to take up prey, specifically fluorescently labeled bacteria (FLB). During the short-term experiments, we estimated grazing rates over FLB for 2 size categories of HNF and MNF. Both bacterial production (BP) and nanophytoplankton primary production (PP) were higher in the river plume area. PNF abundance ranged from 6 × 10 9 to 411 × 10 9 cells m �2 , whereas HNF abundance fluctuated between 27 × 10 9 and 267 × 10 9 cells m �2 . In contrast, the abundance of MNF was usually low, with a maximum of ~7 × 10 9 cells m �2 . For MNF, ingestion rates were between 7.3 and 30.7 bacteria per flagellate and hour (bact flag �1 h �1 ), whereas HNF in- gestion ranged from 2 to 7.5 bact flag �1 h �1 . However, since HNF dominated in terms of abundance, they were the dominant grazers on bacterioplankton populations. Estimates of grazing pressure for the microbial food web showed that MNF were capable of removing 1 to 51% BP d �1 , whereas HNF could control BP, eliminating from 24% BP d �1 up to more than 100% BP d �1 . Given the area's relatively high nutrient condition, the elevated MNF biomass in the river plume and the greater bacterivory impact from MNF in winter, it seems that light and, thus, the energy/carbon limitation could be the main trigger for mixotrophy in this river-influenced coastal upwelling area.

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

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

Will harmful dinoflagellate Karenia mikimotoi grow phagotrophically?

We studied the phagotrophic ability of dinoflagellate strain Karenia mikimotoi KM-Lu (isolated from the South China Sea), using fluorescent microspheres, bacteria isolated from the culture of K. mikimotoi and a marine microalgae Isochrysis galbana. We found that K. mikimotoi cultured under conditions of high light intensity could ingest fluorescent microspheres (diameters 0.5 and 2.0 μm) and fluorescence-labeled bacteria and microalgae. Under a low light intensity, however, only fluorescent microspheres (diameter 0.5 μm) and fluorescence-labeled microalgae were ingested. K. mikimotoi showed better growth by ingesting living marine bacteria or microalgae I. galbana than the controls, either in nutrient-depleted or nutrient-replete conditions. In nutrient-depleted conditions, the growth of K. mikimotoi was more significant with I. galbana as the prey item. In conclusion, the harmful dinoflagellate K. mikimotoi from the South China Sea has apparent phagotrophic ability, and some marine bacteria and microalgae may promote the growth of K. mikimotoi.

Importance of bacterivory by pigmented and heterotrophic nanoflagellates during the warm season in a subtropical western Pacific coastal ecosystem

We investigated temporal variations in the effects of bacterivory by different sizes of heterotrophic nanoflagellates (HNF) and pigmented nanoflagellates (PNF) during a warm period (May to September) in oligotrophic coastal waters of the subtropical western Pacific. Short-term experiments with fluorescently labeled bacteria (FLB) demonstrated ingestion rates of 0.3 to 5.8 bacteria HNF ―1 h ―1 by HNF in the size range 3―6 pm—rates that were higher than observed for other sizes of HNF. Rates of ingestion by PNF ranged between 0.9 and 15.5 cells PNF- 1 h ―1 , and, as for HNF, were greatest for PNF in the 3―6 μm size group. Nanoflagellates of size < 6 μm removed about 98 % of the total amount of bacteria consumed. The 3―6 um PNF, 2―3 um HNF, and 3―6 um HNF were major consumers in the nanoflagellate community and were responsible for an average of 52, 28 and 16% of the total consumption of bacteria, respectively. The smallest PNF (2―3 um) consumed only about 2 % of the total and were considered to be primarily autotrophic. Despite ingestion rates in the range of those reported elsewhere, the low abundance of nanoflagellates observed resulted in relatively low grazing impacts (<10% of bacterial standing stock). We found a significant negative correlation between PO 4 concentrations and ingestion rates of the 3―6 μm PNF, suggesting that the PNF ingestion rate increased under nutrient-deficient conditions.