The cultivation of the rumen ciliate Entodinium bursa in the presence of Entodinium caudatum.

The rumen ciliate protozoon Entodinium bursa has been grown in vitro in the presence of bacteria and Entodinium caudatum for over a year at population densities of 100 to 200 ml-1. The medium contained potassium phosphate, prepared fresh rumen fluid, cysteine, wholemeal flour (or rice starch), dried grass and a culture of the spineless form of Entodinium caudatum. Entodinium bursa has an obligate requirement for this protozoon and died within 48 h in its absence. During growth from a 2% inoculum, the mean generation time of E. bursa was 6 h. Entodinium bursa engulfed 1-5 to 2-5 E. caudatum organisms h-1, and when E. caudatum was in excess it developed caudal spines for the first time in 17 years; these spined forms were engulfed much less readily than the spineless organisms.     

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa

C oleman , G.S. & H all , F.J. 1984. The uptake and utilization of Entodinium caudatum , bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa. Journal of Applied Bacteriology 56 , 283–294.
Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum , ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus mega-terium, Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bouis , although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and i>Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro , although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa . Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.     

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa

Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum, ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus megaterium. Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bovis, although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro, although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa. Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.     

The metabolism of starch, glucose, amino acids, purines, pyrimidines and bacteria by the rumen ciliate Polyplastron multivesiculatum.

The large rumen ciliate protozoon Polyplastron multivesiculatum grown in vitro engulfed a wide range of bacteria (from a population density of 10(9) bacteria ml(-1)) at a rate of 1500 to 137000 bacteria h(-1) protozoon(-1). No evidence was found for the preferential engulfment of bacteria of rumen origin. Except for Proteus mirabilis none of the bacteria were digested with the liberation of soluble materials into the medium. Glucose and amino acids were taken up rapidly by P. multivesiculatum compared with the rate of uptake by Entodinium caudatam. Glucose was incorporated into protozoal polysaccharide and into bacteria associated with the protozoa and was used for the synthesis of a wide range of amino acids. Evidence showed that bacteria and free amino acids at the concentrations found in the rumen could supply the protein requirements of the protozoa for division at least once each day.     

The Uptake and Utilization of Bacteria, Amino Acids and Carbohydrate by the Rumen Ciliate Entodinium longinucleatum in Relation to the Sources of Amino Acids for Protein Synthesis

Entodinium longinucleatum grown in vitro in the presence of bacteria engulfed a wide range of bacterial species at rates of 130–3400 bacteria/h/protozoon (from suspensions of 10 bacteria/ml), but showed a preference for Klebsiella aerogenes and Proteus mirabilis which occurred in the growth medium. Some of the bacteria were digested with release of soluble material into the medium. Free amino acids were incorporated by the protozoa in the presence of chloramphenicol at rates of 5·4–15·1 nmol/h/10⁶ protozoa and approximately 40% of the amino acid-carbon was incorporated into protein. There was no appreciable synthesis of protozoal protein from carbohydrate. Evidence was obtained that the protozoa obtained the amino acids required for growth largely from engulfed bacteria.     

Electron microscopy of the rumen ciliate Entodinium caudatum, with special reference to the engulfment of bacteria and other particulate matter

A study in the electron microscope of thin sections of the rumen ciliate Entodinium caudatum was undertaken in an attempt to elucidate the mode of engulfment of particulate matter. This protozoon engulfed bacteria, polystyrene latex particles and olive oil into membrane-lined vesicles in the protozoal endoplasm. Particles of palladium black were also taken up into the endoplasm, but due to the toxic nature of this material it was not possible to demonstrate vesicle formation with certainty. The initial uptake of bacteria may be into large sacs containing many organisms which were subsequently taken into the endoplasm in vesicles that contained only one bacterium each. The evidence obtained in this investigation has been used to distinguish between two different mechanisms for the digestion of bacteria and utilization of the amino acids from the bacterial protein for the synthesis of protozoal protein.     

The Uptake and Utilization of Bacteria, Amino Acids and Nucleic Acid Components by the Rumen Ciliate Eudiplodinium maggii

Washed suspensions of the rumen ciliate protozoon Eudiplodinium maggii grown in vitro and incubated anaerobically engulfed all the bacteria tested except for Bacteroides ruminicola and Klebsiella aerogenes. There was considerable variation (160–9100 bacteria/h/protozoon at an external concentration of 10¹⁰ bacteria/ml) in the rate at which the bacteria were engulfed, but Eu. maggii showed some preference for bacteria of rumen origin. Some of the bacteria were digested with the release of soluble materials into the medium. Free amino acids were incorporated from an 0.1 mM solution at rates of 0.13 to 0.84 pmol/h/protozoon. Evidence is presented that Eu. maggii could obtain half the amino acids required for growth by the engulfment and digestion of bacteria and half by the uptake of free amino acids. Eudiplodinium maggii incorporated uridine 5' monophosphate and also hydrolysed this to uridine and then to uracil which was reduced to dihydrouracil. These products all appeared in the medium. Ribose was incorporated by the protozoon and appeared as glucose in protozoal and bacterial polysaccharide; none was incorporated as such into protozoal nucleic acid.     

The uptake of bacteria and amino acids by Ophryoscolex caudatus, Diploplastron affine and some other rumen Entodiniomorphid protozoa

The rate of uptake of mixed rumen bacteria and free amino acids by washed suspensions of seven species of rumen ciliate protozoa has been followed. By assuming that the behaviour of these protozoa was the same under these conditions as during growth it was shown that Ophryoscolex caudatus could obtain the amino acids for growth by the engulfment of rumen bacteria. However, all the cellulolytic protozoa studied (Diploplastron affine, Diplodinium anacanthum, Diplodinium anisacanthum, Enoploplastron triloricatum, Eremoplastron bovis and Ostracodinium obtusum bilobum) were unable to obtain sufficient amino acids from either source to grow at even 25% of the maximum rate and it is postulated that they might utilize plant protein. O. caudatus grown in vitro did not engulf Klebsiella aerogenes or Escherichia coli but took up other bacteria and a rumen yeast at rates of up to 54000 organisms/protozoon/h from a population density of 10⁹/ml. When grown in vivo it was more selective and engulfed mixed rumen bacteria at only 10% of the rate obtained with protozoa grown in vitro. D. affine grown in vitro did not engulf Bacteroides ruminicola, Esch. coli, Kl. aerogenes or Proteus mirabilis but took up mixed rumen bacteria from a population of 10⁹/ml at a rate of 2200 bacteria/ protozoon/h.     

The rate of uptake and metabolism of starch grains and cellulose particles by Entodinium species, Eudiplodinium maggii, some other entodiniomorphid protozoa and natural protozoal populations taken from the ovine rumen.

The rates of engulfment and breakdown of starch grains and cellulose particles and of the rate of synthesis of amylopectin from cellulose by individual species of entodiniomorphid protozoa (grown in vivo and in vitro) and incubated anaerobically in vitro were studied. Rates of starch uptake varied from 2.3 to 770 micrograms/mg protozoal protein/min; the lowest was found with Diploplastron affine and the highest with Entodinium spp. on initial incubation with starch grains. The rate of starch breakdown varied from 0.49 to 8.6 micrograms/mg protein/min; the rate was dependent on the initial starch concentration inside the protozoa. Eudiplodinium maggii engulfed cellulose particles more rapidly (2-7 times) than rice starch grains and digested the cellulose at rates of 10 to 16.5 micrograms/mg protein/min. In a mixture of starch grains and cellulose particles, it engulfed the latter at 1.35 to 25 times the rate of the former. Eudiplodinium maggii and Epidinium caudatum, but not Entodinium spp. or Dip. affine, synthesized an amylopectin-like material from cellulose at rates of 0.4 to 4.75 micrograms/mg protein/min. If these reactions occur in the rumen in vivo, up to 9 g of amylopectin could be synthesized from cellulose each day by the entodiniomorphid protozoa.     

The rate of uptake and metabolism of starch grains and cellulose particles by Entodinium species, Eudiplodinium maggii, some other entodiniomorphid protozoa and natural protozoal populations taken from the ovine rumen

The rates of engulfment and breakdown of starch grains and cellulose particles and of the rate of synthesis of amylopectin from cellulose by individual species of entodiniomorphid protozoa (grown in vivo and in vitro ) and incubated anaerobically in vitro were studied. Rates of starch uptake varied from 2.3 to 770 μg/mg protozoal protein/min; the lowest was found with Diploplastron affine and the highest with Entodinium spp. on initial incubation with starch grains. The rate of starch breakdown varied from 0.49 to 8.6 μg/mg protein/min; the rate was dependent on the initial starch concentration inside the protozoa. Eudiplodinium maggii engulfed cellulose particles more rapidly (2–7 times) than rice starch grains and digested the cellulose at rates of 10 to 16.5 μg/mg protein/min. In a mixture of starch grains and cellulose particles, it engulfed the latter at 1.35 to 25 times the rate of the former. Eudiplodinium maggii and Epidinium caudatum , but not Entodinium spp. or Dip. affine , synthesized an amylopectin-like material from cellulose at rates of 0.4 to 4.75 μg/mg protein/min. If these reactions occur in the rumen in vivo , up to 9 g of amylopectin could be synthesized from cellulose each day by the entodiniomorphid protozoa.     

Growth Factors of Bacterial Origin for the Culture of the Rumen Oligotrich Protozoon, Entodinium caudatum

Attempts were made to develop an artificial medium suitable for axenic culture of Entodinium caudatum. Agnotobiotic cultures of the protozoon were established as stock cultures for testing the suitability of various growth media. A cell-free extract of mixed bacteria isolated from the rumen was shown to contain one or more growth factors for the protozoon when supplied with activated charcoal as a carrier. The medium (CYSE medium), which supported the growth of the protozoon in the presence of 50 μg/ml each of penicillin and chloramphenicol, consisted of activated charcoal (20 mg), heat-treated yeast (Y) (80 mg), 13%β-sitosterol-coated rice starch (S) (120 mg), and cell-free extract of rumen bacteria (1 ml) in 40 ml buffer solution. When culturing the protozoon, the CYSE medium was supplemented daily with 20 mg each of Y and S and half of the medium was replaced with fresh medium once every 5 d. The possible use of this method to establish an axenic culture of E. caudatum is discussed.     

The reversible effect of flow on the morphology of ceratocorys horrida (PERIDINIALES,DINOPHYTA)*

Most cells experience an active and variable fluid environment, in which hydrodynamic forces can affect aspects of cell physiology including gene regulation, growth, nutrient uptake, and viability. The present study describes a rapid yet reversible change in cell morphology of the marine dinoflagellate Ceratocorys horrida Stein, due to fluid motion. Cells cultured under still conditions possess six large spines, each almost one cell diameter in length. When gently agitated on an orbital shaker under conditions simulating fluid motion at the sea surface due to light wind or surface chop, as determined from digital particle imaging velocimetry, population growth was inhibited and a short‐spined cell type appeared that possessed a 49% mean decrease in spine length and a 53% mean decrease in cell volume. The reduction in cell size appeared to result primarily from a 39% mean decrease in vacuole size. Short‐spined cells were first observed after 1 h of agitation at 20°C; after 8 to 12 d of continuous agitation, long‐spined cells were no longer present. The morphological change was completely reversible; in previously agitated populations devoid of long‐spined cells, cells began to revert to the long‐spined morphology within 1 d after return to still conditions. During morphological reversal, spines on isolated cells grew up to 10 μm·d^?1. In 30 d the population morphology had returned to original proportions, even though the overall population growth was zero during this time. The reversal did not occur as a result of cell division, because single‐cell studies confirmed that the change occurred in the absence of cell division and much faster than the 16‐d doubling time. The threshold level of agitation causing morphology change in C. horrida was too low to inhibit population growth in the shear‐sensitive dinoflagellate Lingulodinium polyedrum. At the highest level of agitation tested, there was negative population growth in C. horrida cultures, indicating that fluid motion caused cell mortality. Small, spineless cells constituted a small percentage of the population under all conditions. Although their abundance did not change, single‐cell studies and morphological characteristics suggest that the spineless cells can rapidly transform to and from other cell types. The sinking rate of individual long‐spined cells in still conditions was significantly less than that of short‐spined cells, even though the former are larger and have a higher cell density. These measurements demonstrate that the long spines of C. horrida reduce cell sinking. Shorter spines and reduced swimming would allow cells to sink away from turbulent surface conditions more rapidly. The ecological importance of the morphological change may be to avoid conditions that inhibit population growth and potentially cause cell damage.     

More monensin-sensitive, ammonia-producing bacteria from the rumen

Two monensin-sensitive bacteria which utilized carbohydrates poorly and grew rapidly on amino acids were isolated from the bovine rumen. The short rods (strain SR) fermented arginine, serine, lysine, glutamine, and threonine rapidly (greater than 158 nmol/mg of protein per h) and grew faster on casein digest containing short peptides than on free amino acids ().34 versus 0.29 h(-1)). Gelatin hydrolysate, an amino acid source containing an abundance of long peptides, was unable to support growth or ammonia production, but there was a large increase in ammonia production if strain SR was cocultured with peptidase-producing ruminal bacteria (Bacteroides ruminicola or Streptococcus bovis). Cocultures showed no synergism with short peptides. Strain SR washed out of continuous culture ().1 h(-1)) at pH 5.9. The irregularly shaped organisms (strain F) deaminated glutamine, histidine, glutamate, and serine rapidly (greater than 137 nmol/mg of protein per min) and grew faster on free amino acids than on short peptides ().43 versus 0.21 h(-1)). When strain F was provided with casein or gelatin hydrolysate and cocultured with peptidase-producing bacteria, there was a more than additive increase in ammonia production. Strain F grew in continuous culture (0.1 h(-1)) when the pH was as low as 5.3. The irregularly shaped cells and short rods were present at less than 10(9)/ml in vivo, but they ahd very high specific activities of ammonia production (greater than 310 nmol of ammonia/mg of protein per min) and could play an important role in ruminal amino acid fermentation.     

More monensin-sensitive, ammonia-producing bacteria from the rumen.

Two monensin-sensitive bacteria which utilized carbohydrates poorly and grew rapidly on amino acids were isolated from the bovine rumen. The short rods (strain SR) fermented arginine, serine, lysine, glutamine, and threonine rapidly (greater than 158 nmol/mg of protein per h) and grew faster on casein digest containing short peptides than on free amino acids ().34 versus 0.29 h(-1)). Gelatin hydrolysate, an amino acid source containing an abundance of long peptides, was unable to support growth or ammonia production, but there was a large increase in ammonia production if strain SR was cocultured with peptidase-producing ruminal bacteria (Bacteroides ruminicola or Streptococcus bovis). Cocultures showed no synergism with short peptides. Strain SR washed out of continuous culture ().1 h(-1)) at pH 5.9. The irregularly shaped organisms (strain F) deaminated glutamine, histidine, glutamate, and serine rapidly (greater than 137 nmol/mg of protein per min) and grew faster on free amino acids than on short peptides ().43 versus 0.21 h(-1)). When strain F was provided with casein or gelatin hydrolysate and cocultured with peptidase-producing bacteria, there was a more than additive increase in ammonia production. Strain F grew in continuous culture (0.1 h(-1)) when the pH was as low as 5.3. The irregularly shaped cells and short rods were present at less than 10(9)/ml in vivo, but they ahd very high specific activities of ammonia production (greater than 310 nmol of ammonia/mg of protein per min) and could play an important role in ruminal amino acid fermentation.     

Morphological variation of <Emphasis Type="Italic">Keratella cochlearis</Emphasis> (Gosse) in Myanmar (Burma) in relation to zooplankton community structure

Keratella cochlearis was present in 27 of 35 water bodies sampled in Myanmar, and was the most abundant rotifer in 10. Measurements of lorica length and posterior spine length from 20 localities showed that posterior spine length varied both with lorica length and with the composition of the crustacean zooplankton. Long spines were associated with dominance by Heliodiaptomus. The shortest spines were found in samples dominated by cladocerans or cyclopoid copepods. Posterior spine length was positively correlated with the number of diaptomid copepods. Forms without posterior spines were found in 17 localities. The lorica lengths of these spineless forms were generally similar to those of co-occurring spined forms (r = 0.68), but in a few samples the loricas of the spineless forms were significantly larger. These larger forms are similar to the ‘aspina’ forms recently recognised in the River Thames in England. These samples were dominated by cladocerans or cyclopoid copepods. In one locality spineless forms were found without spined forms. The crustacean zooplankton in this locality was also dominated by cladocerans. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont & R. Rico-Martínez Advances in Rotifer Research     

Acanthodasys caribbeanensis sp. n., a new species of Thaumastodermatidae (Gastrotricha, Macrodasyida) from Belize and Panama

We describe one new species of Acanthodasys (Gastrotricha, Macrodasyida, Thaumastodermatidae) collected from sublittoral sites around Carrie Bow Cay, Belize and Isla Colón in the Bocas del Toro archipelago, Panama. Though eight species of Acanthodasys are currently recognized, no species has yet been reported from the Caribbean. Acanthodasys caribbeanensissp. n. is characterized by the lack of lateral adhesive tubes, the presence of ventrolateral adhesive tubes, and with cuticular armature in the form of both spineless and spined scales. The spineless scales are not elliptical as in other species of Acanthodasys, but are instead variable in shape and closely resemble the spineless scales of species of Diplodasys. Spined scales bear uniancres up to 50 μm long and are the largest reported in the genus. Uniancres are arranged dorsally around the mouth rim and distributed in five distinguishable columns. Adult size varies from 325-625 μm long.     

Interactions of Tetrahymena pyriformis, Escherichia coli, Azotobacter vinelandii, and Glucose in a Minimal Medium

A study was made of the food web formed from a protozoon, two bacteria, and a glucose minimal medium in chemostat culture. The system was also divided into simpler parts, first by omitting the protozoon to obtain a competition system, and then by omitting one or the other of the bacteria to obtain two food chains. In the competition studies, one bacterium was displaced by the other at all holding times used. In the food chain studies, sustained oscillations of the population densities of predator and prey developed at short holding times, and then changed to damped oscillations at longer holding times. In addition, the level of residual glucose remained high at long holding times. A new model of microbial growth is necessary to explain these results. In the food web studies, predation of the protozoon on the two bacteria stabilized the competition between the latter and allowed their coexistence in the same habitat. Thus, Gause's principle was circumvented.     

Suitability of RAPD for analyzing spined and spineless variant regenerants of pineapple (Ananas comosus L., Merr.)

A random amplified polymorphic DNA (RAPD) analysis of spineless (variant phenotype) plants obtained from micropropagated dormant pineapple (Ananas comosus L., Merr.) axillary buds was performed using arbitrary 10-mer oligonucleotide primers. This was done to investigate the genetic fidelity of the regenerants and to distinguish these variants from regenerants bearing the normal spined phenotype. Of the 58 arbitrary primers used, 29 produced bands unique to the spineless phenotype, and 30 produced bands unique to the spined phenotype. A total of 914 bands were scored, 55 of which were polymorphic to the spineless phenotype and 51 of which were polymorphic to the spined phenotype. On the basis of RAPD amplification products, genetic similarity was estimated in both types of regenerants using similarity coefficients (Nei and Li, 1979). The characteristic finger-prints generated by each probe emphasize genetic variability of regenerants. This technique is suitable for analyzing variant regenerants induced in vitro.     

Relationships Among Deoxyribonucleic Acid, Ribonucleic Acid, and Specific Transfer Ribonucleic Acids in Escherichia coli 15T− at Various Growth Rates

The levels of macromolecules in Escherichia coli 15T(-) growing in broth, glucose, succinate, and acetate media were determined to compare relationships among deoxyribonucleic acid (DNA), ribosomal ribonucleic acid (rRNA), transfer RNA (tRNA), and protein in cells at different growth rates. DNA and protein increased in relative amounts with decreasing growth rate; relative amounts of rRNA and tRNA decreased, tRNA making up a slightly larger proportion of RNA. For several amino acid-specific tRNAs studied, acceptor capacities per unit of DNA increased with increasing growth rate. The syntheses of tRNA and rRNA are regulated by similar, yet different, mechanisms. Chromatographic examination on columns of benzoylated diethylaminoethyl-cellulose of isoaccepting tRNAs for arginine, leucine, lysine, methionine, phenylalanine, serine, and valine did not reveal differences in the isoaccepting profiles for rapidly (broth culture) and slowly growing (acetate culture) cells. Therefore, isoacceptors for individual amino acids appear to be regulated as a group. Lower efficiencies of ribosomal function in protein synthesis can be explained, in part, by a low ratio of tRNA to the number of ribosomes available and by a decreasing concentration of tRNA with decreasing growth rate. Data on the tRNAs specific for seven amino acids indicate that the decreasing concentration of tRNA is a general event rather than a severe limitation of any one tRNA or isoaccepting tRNA.     

Interactions between bacteria-feeding nematodes and bacteria in the rape rhizosphere: effects on root exudation and distribution of bacteria

Abstract Effects of rhizosphere bacteria (RB), and rhizosphere bacteria with bacteria-feeding nematodes (RBN), on the composition of root exudate were examined after 2 weeks in gnotobiotic culture systems with rape seedlings ( Brassica napus (L.)). The amounts of low molecular weight carbohydrates and of some free amino acids, per unit root dry weight, in the exudates were lower in the RB and RBN treatments than in the axenic control (R treatment). The growth of nematodes implied a production of bacterial cells in the RBN treatment 2.6 times that in the RB treatment. The bacterial growth in the RB treatment and the bacterial growth in combination with grazing by nematodes in the RBN treatment implied 24 and 63 times as much exudation of organic carbon, respectively, as in the R treatment.
Most bacteria were attached to sand particles. The nematodes being suspension-feeders, decreased the proportion of free bacteria from 6% in the RB treatment to 2% in the RBN treatment. The numbers of attached bacteria in the RBN treratment were positively correlated with the numbers of nematodes, indicating stimulation of bacterial growth by the grazing.