Differential grazing of two heterotrophic nanoflagellates on marine Synechococcus strains

Grazing of heterotrophic nanoflagellates on marine picophytoplankton presents a major mortality factor for this important group of primary producers. However, little is known of the selectivity of the grazing process, often merely being thought of as a general feature of cell size and motility. In this study, we tested grazing of two heterotrophic nanoflagellates, Paraphysomonas imperforata and Pteridomonas danica , on strains of marine Synechococcus . Both nanoflagellates proved to be selective in their grazing, with Paraphysomonas being able to grow on 5, and Pteridomonas on 11, of 37 Synechococcus strains tested. Additionally, a number of strains (11 for Paraphysomonas , 9 for Pteridomonas ) were shown to be ingested, but not digested (and thus did not support growth of the grazer). Both the range of prey strains that supported growth as well as those that were ingested but not digested was very similar for the two grazers, suggesting a common property of these prey strains that lent them susceptible to grazing. Subsequent experiments on selected Synechococcus strains showed a pronounced difference in grazing susceptibility between wild-type Synechococcus sp. WH7803 and a spontaneous phage-resistant mutant derivative, WH7803PHR, suggesting that cell surface properties of the Synechococcus prey are an important attribute influencing grazing vulnerability.     

Effects of bacterial prey species and their concentration on growth of the amoebae Acanthamoeba castellanii and Hartmannella vermiformis

Two amoebae were presented with six bacterial prey at a range of concentrations, and the growth parameters of the amoebae were deduced. All but one bacterium (Synechococcus) resulted in a positive growth response, but the gram-positive bacterium Staphylococcus aureus proved to be difficult to digest and the heavily pigmented bacterium Klebsiella ozaenae induced unusual amoebic behavior prior to ingestion.     

The Feeding of Amoeba proteus on Paramecium aurelia*

SYNOPSIS. Density of prey (Paramecium aurelia) and predator (Amoeba proteus) were varied while volume of inorganic medium was kept constant. Variations in density of prey had little effect on the rates of feeding and reproduction of the amoebae; but with increasing predator density the amoebae captured the paramecia less rapidly and ingested fewer before dividing, altho division size did not change appreciably. Therefore, amoebae of a low density population with a constant food supply carry more nutritional reserves from generation to generation than do those in a denser population.     

Testate amoebae (Protista) communities in Hylocomium splendens (Hedw.) B.S.G. (Bryophyta): relationships with altitude, and moss elemental chemistry

We studied the testate amoebae in the moss Hylocomium splendens along an altitudinal gradient from 1000 to 2200 m asl. in the south-eastern Alps of Italy in relation to micro- and macro-nutrient content of moss plants. Three mountainous areas were chosen, two of them characterised by calcareous bedrock, the third by siliceous bedrock. A total of 25 testate amoebae taxa were recorded, with a mean species richness of 9.3 per sampling plot. In a canonical correspondence analysis, 63.1% of the variation in the amoebae data was explained by moss tissue chemistry, namely by C, P, Ca, Mg, Al, Fe, and Na content and a binary site variable. We interpreted this result as an indirect effect of moss chemistry on testate amoebae through an influence on prey organisms. Although two species responded to altitude, there was no overall significant relationship between testate amoebae diversity or community structure and altitude, presumably because our sampling protocol aimed at minimizing the variability due to vegetation types and soil heterogeneity. This suggests that previous evidence of altitudinal or latitudinal effects on testate amoebae diversity may at least in part be due to a sampling bias, namely differences in soil type or moss species sampled.     

Selection of cyanobacteria isolated from mosquito breeding sites as a potential food source for mosquito larvae.

One way to increase the persistence of larvicidal toxins in mosquito breeding sites is to clone the corresponding genes in microorganisms, such as cyanobacteria, which could serve as a source of food for the larvae. We isolated and cultured 10 strains of cyanobacteria from three mosquito breeding sites along the French Mediterranean coast. Most of the strains were tolerant to a relatively wide range of salt concentrations, and all of them were totally or partially resistant to at least four of the five biological or chemical larvicides used in the local mosquito control program. Six unicellular strains from these habitats and Synechococcus strain PCC 7942, a strain maintained for more than 10 years under laboratory conditions, were assessed for ingestion and digestion by larvae Culex pipiens and Anopheles gambiae mosquitoes. The numbers of cells ingested and digested were dependent on the cyanobacterial strain and varied with the mosquito species. Three of the new isolates, Synechococcus strain PCC 8905 and Synechocystis strains PCC 8906 and PCC 8912, were ingested and digested rapidly by larvae of both mosquito species. Since these strains are also tolerant to larvicides and relatively resistant to elevated salt concentrations, they meet the basic requirements for potential recipients of bacterial genes that encode endotoxins.     

Selection of cyanobacteria isolated from mosquito breeding sites as a potential food source for mosquito larvae.

One way to increase the persistence of larvicidal toxins in mosquito breeding sites is to clone the corresponding genes in microorganisms, such as cyanobacteria, which could serve as a source of food for the larvae. We isolated and cultured 10 strains of cyanobacteria from three mosquito breeding sites along the French Mediterranean coast. Most of the strains were tolerant to a relatively wide range of salt concentrations, and all of them were totally or partially resistant to at least four of the five biological or chemical larvicides used in the local mosquito control program. Six unicellular strains from these habitats and Synechococcus strain PCC 7942, a strain maintained for more than 10 years under laboratory conditions, were assessed for ingestion and digestion by larvae Culex pipiens and Anopheles gambiae mosquitoes. The numbers of cells ingested and digested were dependent on the cyanobacterial strain and varied with the mosquito species. Three of the new isolates, Synechococcus strain PCC 8905 and Synechocystis strains PCC 8906 and PCC 8912, were ingested and digested rapidly by larvae of both mosquito species. Since these strains are also tolerant to larvicides and relatively resistant to elevated salt concentrations, they meet the basic requirements for potential recipients of bacterial genes that encode endotoxins.     

Functional and aggregative response of North Sea whiting

The functional response of whiting (Merlangius merlangus L.) to clupeid and gadoid prey was determined from estimates of food intake and prey density at five locations in the North Sea. The intake of most prey types was well described by a type II (decelerating) response, although in some cases a type III (sigmoid) response provided a slightly better fit. Though a saturation level was reached for all types of fish prey, none of the levels corresponded to the maximum digestive capacity of the predator. This was not caused by ingestion of other prey as the amount of other food and fish prey ingested were not negatively correlated. An investigation of the occurrence of fresh fish in the stomachs revealed that fish was ingested almost exclusively during dawn and dusk and the lack of negative correlation between the intake of fish and other prey may thus be a result of the limited time in which fish prey was vulnerable to predation. No aggregative response of the predators was detected towards any of the prey and catches of prey and predators were slightly negatively correlated. There was evidence of an increase in mortality with density at low clupeid densities, but mortality decreased to virtually zero at high densities. Whiting seem therefore unlikely to impose a regulatory effect on their fish prey outside a narrow range of prey densities.     

ENGULFMENT AND ANASTOMOSIS IN THE CELLULAR SLIME MOLDS (ACRASIALES)

Evidence for engulfment and anastomosis of the myxamoebae of several members of the Acrasiales is presented. Photomicrographic evidence for engulfment and anastomosis indicates that these processes are unrelated to any regular sexual mechanism. Vacuolar activity was found to be associated with plasma membrane properties in both anastomosis and engulfment. Frequent multiple engulfment, progressive destruction and digestion of the ingested cells, and ultimate egestion of both intact and partially digested engulfed cells are interpreted as evidence favoring the view that engulfment is a cannibalistic activity. Binucleate and multinucleate amoebae were produced by cell fusion; however, no supporting evidence for nuclear fusion was obtained from observations of either anastomosis or engulfment. It is concluded that no regular sexuality occurs in the usual life cycle of the Acrasiales.     

Feeding Characteristics of an Amoeba (Lobosea: Naegleria) Grazing Upon Cyanobacteria: Food Selection, Ingestion and Digestion Progress

Bacterivory by heterotrophic nanoflagellates and ciliates has been widely studied in aquatic environments, but data on the grazing of amoebae, are still scarce. From the water samples of Dianchi Lake (Kunming, Yunnan Province, China), we isolated an amoeba, designated as Naegleria sp. strain W2, which had potent grazing effects on some kind of cyanobacteria. The food selection mechanism and the digestion process of the amoeba were investigated in batch experiments. Predation experiments showed that filamentous cyanobacteria (e.g., Anabaena, Cylindrospermum, Gloeotrichia, and Phormidium) were readily consumed, with clearance rates ranging from 0.332 to 0.513 nL amoeba⁻¹ h⁻¹. The tight threads (Oscilltoria) and aggregates (Aphanizomenon) could not be ingested; however, their sonicated fragments were observed inside food vacuoles, suggesting that their morphologies prevent them from being ingested. Live video microscopy noted that unicellular Chroococcaceae (e.g., Synechococcus, Aphanocapsa, and Microcystis) were excreted after ingestion, indicating that food selection takes place inside food vacuoles. To determine whether the tastes or the toxins prevented them from being digested, heat-killed cells were retested for predation. Digestion rates and ingestion rates of the amoebae for filamentous cyanobacteria were estimated from food vacuole content volume. Through a “cold-chase” method, we found that the food vacuole contents declined exponentially in diluted amoebae cells, and digestion rates were relatively constant, averaging about 1.5% food vacuole content min⁻¹ at 28°C. Ingestion strongly depended on the satiation status of the amoebae, starved amoebae fed at higher rates compared with satiated amoebae. Our results suggest that the food selection and food processing mechanisms of the amoeba are similar to those of interception feeding flagellates; however, filamentous cyanobacteria cannot obtain a refuge under the grazing pressure of phagotrophic amoebae, which may widen our knowledge on the grazing of protists.     

Confusing Selective Feeding with Differential Digestion in Bacterivorous Nanoflagellates

Food selectivity and the mechanisms of food selection were analyzed by video microscopy for three species (Spumella, Ochromonas, Cafeteria) of interception-feeding heterotrophic nanoflagellates. The fate of individual prey particles, either live bacteria and/or inert particles, was recorded during the different stages of the particle-flagellate-interaction, which included capture, ingestion, digestion, and egestion. The experiments revealed species-specific differences and new insights into the underlying mechanisms of particle selection by bacterivorous flagellates. When beads and bacteria were offered simultaneously, both particles were ingested unselectively at similar rates. However, the chrysomonads Spumella and Ochromonas egested the inert beads after a vacuole passage time of only 2-3 min, which resulted in an increasing proportion of bacteria in the food vacuoles. Vacuole passage time for starved flagellates was significantly longer compared to that of exponential-phase flagellates for Spumella and Ochromonas. The bicosoecid Cafeteria stored all ingested particles, beads as well as bacteria, in food vacuoles for more then 30 min. Therefore "selective digestion" is one main mechanism responsible for differential processing of prey particles. This selection mechanism may explain some discrepancies of former experiments using inert particles as bacterial surrogates for measuring bacterivory.     

Interactions of some common pathogenic bacteria with Acanthamoeba polyphaga

Protozoan grazing is a major trophic pathway whereby the biomass re-enters the food web. Nonetheless, not all bacteria are digested by protozoa and the number known to evade digestion, resulting in their environmental augmentation, is increasing. We investigated the interactions of Bacillus cereus, Enterococcus faecalis, Enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and methicillin-sensitive Staphylococcus aureus (MSSA), with the amoeba, Acanthamoeba polyphaga. There was evidence of predation of all bacterial species except L. monocytogenes and S. aureus, where extracellular numbers were significantly higher when cultured with amoebae compared with growth in the absence of amoebae. Intracellular growth kinetic experiments and fluorescent confocal microscopy suggest that S. aureus survived and may even multiply within A. polyphaga, whereas there was no apparent intra-amoebal replication of L. monocytogenes and higher numbers were likely sustained on metabolic waste products released during coculture.     

Acanthamoeba castellanii: structural basis of the cytopathic mechanisms

In this study we report observations on the structural mechanisms of the cytopathic effect of Acanthamoeba castellanii trophozoites on cultured MDCK cell monolayers. Co-incubations were carried out for a maximum of 24h. The first evidence of damage to the cell monolayer was detected by measuring the transepithelial resistance of cell monolayers that interacted with the amoebae. At 6h, transepithelial resistance diminished to 51% and amoebae required 5-6h to produce evidence of structural injury at the light microscopy level. Following 12h of incubation, the cell monolayer was severely damaged. After making intimate contact with the surface of target cells, trophozoites detached cells from the substrate, lysed and by means of food-cups ingested the damaged cells. There was no morphological evidence of modifications in MDCK cell membranes, membrane fusion or junction formation between the amoeba and host plasma membrane. The lytic capacity of the amoebas appears to be the result of cytotoxic factors secreted by the amoebae since, when monolayers were incubated with conditioned medium, there was also a decrease in the transepithelial resistance. Besides, mechanical injury produced by the attachment and movement of the trophozoites may contribute to the disruption of the cell monolayer. As in other pathogenic amoebae, the cytopathic action of A. castellanii on the cell monolayers can subjectively be separated into four stages: adhesion, cytolysis, phagocytosis, and intracellular degradation.     

A giant cell surface protein in Synechococcus WH8102 inhibits feeding by a dinoflagellate predator

Diverse strains of the marine planktonic cyanobacterium Synechococcus sp. show consistent differences in their susceptibility to predation. We used mutants of Sargasso Sea strain WH8102 (clade III) to test the hypothesis that cell surface proteins play a role in defence against predation by protists. Predation rates by the heterotrophic dinoflagellate Oxyrrhis marina on mutants lacking the giant SwmB protein were always higher (by 1.6 to 3.9×) than those on wild-type WH8102 cells, and equalled predation rates on a clade I strain (CC9311). In contrast, absence of the SwmA protein, which comprises the S-layer (surface layer of the cell envelope that is external to the outer membrane), had no effect on predation by O. marina. Reductions in predation rate were not due to dissolved substances in Synechococcus cultures, and could not be accounted for by variations in cell hydrophobicity. We hypothesize that SwmB defends Synechococcus WH8102 by interfering with attachment of dinoflagellate prey capture organelles or cell surface receptors. Giant proteins are predicted in the genomes of multiple Synechococcus isolates, suggesting that this defence strategy may be more general. Strategies for resisting predation will contribute to the differential competitive success of different Synechococcus groups, and to the diversity of natural picophytoplankton assemblages.     

Growth of Acanthamoeba castellanii and Hartmannella vermiformis on live, heat-killed and DTAF-stained bacterial prey

The growth responses of two species of amoeba were evaluated in the presence of live, heat-killed and heat-killed/5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF)-stained cells of Escherichia coli, Pseudomonas aeruginosa, Klebsiella aerogenes, Klebsiella ozaenae and Staphylococcus aureus. The specific growth rates of both species were significantly higher with live bacterial prey, the only exception being Hartmannella vermiformis feeding on S. aureus, for which growth rates were equivalent on all prey states. There was no significant difference between growth rates, yield or ingestion rates of amoebae feeding on heat-killed or heat-killed/stained bacterial cells, suggesting that it was the heat-killing process that influenced the amoeba-bacteria interaction. Pretreatment of prey cells had a greater influence on amoebic processing of Gram-negative bacteria compared with the Gram-positive bacterium, which appeared to be as a result of the former cells being more difficult to digest and/or losing their ability to deter amoebic ingestion. These antipredatory mechanisms included microcolony formation in P. aeruginosa, toxin production in K. ozaenae, and the presence of an intact capsule in K. aerogenes. E. coli and S. aureus did not appear to possess an antipredator mechanism, although intact cells of the S. aureus were observed in faecal pellets, suggesting that any antipredatory mechanism was occurring at the digestion stage.     

Immunological determination of digestive rates in the syntopic scorpions Urodacus armatus Pocock and Urodacus novaehollandiae Peters

Scorpions have lengthy periods of inactivity, which may be caused either by a long physiological digestion time or predator avoidance or both. We used a quantitative immunological assay to monitor the amount of prey antigen remaining in the hepatopancreas of Urodacus armatus and U. novaehollandiae for 32 days after a meal to test the hypothesis of a long digestion time. In both species, prey antigen concentration in the hepatopancreas increased between 0.25 and 0.5 days after feeding, probably because of incomplete transfer of prey proteins from the large preoral cavity to the hepatopancreas at this stage of digestion. This was followed by a rapid decline, and by 3 days after feeding less than 25% of ingested antigen remained in each species. Small prey residues could still be detected up to 32 days. Prey decay curves in both species were best fitted by a log-log transformation, and were not significantly different. Since digestion is essentially complete after 3 days, it appears that longer periods of post-feeding inactivity are for predator avoidance, not digestion. However, the 32-day detection period exceeds those reported for other invertebrates, and suggests an unusual digestive physiology in scorptions.     

The Influence of Preculture Conditions and Food Quality on the Ingestion and Digestion Process of Three Species of Heterotrophic Nanoflagellates

The influence of prey characteristics such as motility and size as well as of predator characteristics such as satiation and preculturing diet on the feeding process of interception feeding heterotrophic nanoflagellates was investigated. Three species of gram-negative bacteria, one species of gram-positive bacteria, two species of cyanobacteria (Synechococcus) and inert latex particles were fed as prey particles for three species of heterotrophic nanoflagellates (Spumella, Ochromonas, Cafeteria). Ingestion rates depended on the satiation of the flagellates and especially on the filling status of the food vacuoles. In addition, the ingestion rates depended on the characteristics of the food particle and were modified by pre-culturing the flagellates on either Pseudomonas putida or Bacillus subtilis. Digestion was found to be particle-specific. Cyanobacteria were excreted a few minutes after ingestion whereas heterotrophic bacteria were stored and digested in the food vacuoles. The spectrum of ingested particles is not identical to that of digested particles and thus neither the diet of the flagellates nor their impact on bacterial communities can be calculated simply from food vacuole content. "Selective digestion" could be shown to be an important selection mechanism concerning natural food particles. The digestion strategies of Cafeteria on the one hand and Spumella and Ochromonas on the other hand may be an important factor to explain protozoan species composition and succession in the field. In addition to bacterial abundance and grazing pressure by metazooplankton, the bacterial speciescomposition as well as biochemical variations within bacterial species may influence protozoan species composition and abundance.     

Food selection by bacterivorous protists: insight from the analysis of the food vacuole content by means of fluorescence in situ hybridization

A modified fluorescence in situ hybridization (FISH) method was used to analyze bacterial prey composition in protistan food vacuoles in both laboratory and natural populations. Under laboratory conditions, we exposed two bacterial strains (affiliated with beta- and gamma-Proteobacteria -- Aeromonas hydrophila and Pseudomonas fluorescens, respectively) to grazing by three protists: the flagellates Bodo saltans and Goniomonas sp., and the ciliate Cyclidium glaucoma. Both flagellate species preferably ingested A. hydrophila over P. fluorescens, while C. glaucoma showed no clear preferences. Differences were found in the digestion of bacterial prey with B. saltans digesting significantly faster P. fluorescens compared to two other protists. The field study was conducted in a reservoir as part of a larger experiment. We monitored changes in the bacterial prey composition available compared to the bacteria ingested in flagellate food vacuoles. Bacteria detected by probe HGC69a (Actinobacteria) and R-BT065 were negatively selected by flagellates. Bacteria detected by probe CF319a were initially positively selected but along with a temporal shift in bacterial cell size, this trend changed to negative selection during the experiment. Overall, our analysis of protistan food vacuole content indicated marked effects of flagellate prey selectivity on bacterioplankton community composition.     

Feeding ecology of the lizard Tropidurus oreadicus Rodrigues 1987 (Tropiduridae) at Serra dos Carajás, Pará state, northern Brazil.

Tropidurus species commonly prey on arthropods, but they may also feed on vertebrates and plant material. The lizard Tropidurus oreadicus (Tropiduridae) is common in open vegetation habitats and generally has sexual dimorphism. In this study we analyzed the diet of T. oreadicus at Serra dos Carajás, Pará, in the north of Brazil. Snout-vent length (SVL) and jaw width (JW) were taken for 34 lizards. There was a significant difference in SVL and in JW, with males being larger than females. All lizards analyzed contained food in their stomachs. The diet of T. oreadicus at Serra dos Carajás was characterized by the consumption of a relative wide spectrum of food item categories (21 types of items), consisting of arthropods, part of one vertebrate and plant material, which characterizes the diet of a generalist predator. Volumetrically, the most important items in the diet of both sexes of T. oreadicus were flowers (M = 61.7%; F = 33%) and orthopterans (M = 1.7%; F = 3.5%). Ants were the most frequently consumed (100% for both sexes) and the most numerous (M = 94.5%; F = 89.4%) food item. Flowers also were frequently consumed (M = 91.7%; F = 54.5%), with their relative consumption differing significantly between sexes. There was not a significant sexual difference in prey volume, neither in number of preys per stomach, nor in type of prey ingested. There was no relationship between lizard jaw width and the mean volume of prey. The data showed that T. oreadicus is a relatively generalist lizard in terms of diet and that consumes large volumes of plant material, especially flowers of one species of genus Cassia.     

Survival of coliforms and bacterial pathogens within protozoa during chlorination.

The susceptibility of coliform bacteria and bacterial pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa. Viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory strains of Acanthamoeba castellanii and Tetrahymena pyriformis. Cocultures of bacteria and protozoa were incubated to facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated to release intracellular bacteria. Qualitative susceptibility of protozoan strains to free chlorine was also assessed. Protozoa were shown to survive and grow after exposure to levels of free chlorine residuals that killed free-living bacteria. Ingested coliforms Escherichia coli, Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella oxytoca and bacterial pathogens Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, Legionella gormanii, and Campylobacter jejuni had increased resistance to free chlorine residuals. Bacteria could be cultured from within treated protozoans well after the time required for 99% inactivation of free-living cells. All bacterial pathogens were greater than 50-fold more resistant to free chlorine when ingested by T. pyriformis. Escherichia coli ingested by a Cyclidium sp., a ciliate isolated from a drinking water reservoir, were also shown to be more resistant to free chlorine. The mechanism that increased resistance appeared to be survival within protozoan cells. This study indicates that bacteria can survive ingestion by protozoa. This bacterium-protozoan association provides bacteria with increased resistance to free chlorine residuals which can lead to persistence of bacteria in chlorine-treated water. We propose that resistance to digestion by predatory protozoa was an evolutionary precursor of pathogenicity in bacteria and that today it is a mechanism for survival of fastidious bacteria in dilute and inhospitable aquatic environments.     

Determination of the active portion of the folic acid molecule in cellular slime mold chemotaxis.

From earlier work it is known that folic acid attracts the amoebae of various species of cellular slime molds (11). Here we have tested a wide variety of pteridines, pyrimidines, and pyrazines to determine what part of the folic acid molecule is chemotactically active. It was shown that the activity lies in the pteridine ring itself. Furthermore, the cell-free supernatants of slime mold amoebae contain an enzyme that renders pterin and folic acid chemotactically inactive, which apparently increases the chemotactic sensitivity of the amoebae to those compounds. Despite the fact that slime mold amoebae secrete small amounts of folic acid-related compounds, there is no evidence that folates are acrasins; rather it is postulated that attraction to folates may be a food-seeking device for the amoebae which prey on folate-secreting bacteria in the soil.