Axenic Cultivation of the Cellulolytic Flagellate Trichomitopsis termopsidis (Cleveland) from the Termite Zootermopsis*

SYNOPSIS. Trichomitopsis termopsidis (Cleveland), a cellulolytic hindgut symbiote of the termite Zootermopsis, has been cultivated axenically under anaerobic conditions. The medium consists of cellulose, reduced glutathione, fetal calf serum, yeast extract, and autoclaved rumen fluid or autoclaved rumen bacteria, in a buffered salt solution the composition of which is based on an analysis of Zootermopsis hindgut fluid. The hindgut contents of surface-sterilized termites were inoculated into anaerobic buffer-containing cellulose and serum. Repeated passages yielded mixed cultures of T. termopsidis and termite hindgut bacteria. Flagellates were then inoculated into complete medium containing antibiotics, and after 2 passages, axenic cultures of T. termopsidis were obtained. Various nutritional supplements, including clarified rumen fluid or heat-killed bacteria of several known species failed to support the growth of T. termopsidis when substituted for autoclaved rumen fluid. The flagellates did not grow when any of several carbohydrates were substituted for cellulose. Electron microscopy of flagellates from axenic cultures revealed that cellulose particles and partially digested bacteria were present in food vacuoles. No endosymbiotic bacteria were present in the cytoplasm indicating that T. termopsidis does not depend on living prokaryotes for cellulose digestion. The results suggest that T. termopsidis possesses the enzyme cellulase.     

Nutrition and Growth Characteristics of Trichomitopsis termopsidis, a Cellulolytic Protozoan from Termites

Putatively axenic cultures of Trichomitopsis termopsidis 6057, isolated by M. A. Yamin (J. Protozool. 25:535-538, 1978) from the hindgut of Zootermopsis termites, apparently contained methanogenic bacteria, inasmuch as small amounts of CH₄ were produced during growth. However, T. termopsidis could be “cured” of methanogenic activity by incubation in the presence of bromoethanesulfonate. Both the cured derivative (6057C) and the parent strain (6057) required NaHCO₃ and fetal bovine serum for good growth; the presence of yeast extract in media was stimulatory. Growth of both strains was markedly improved by substituting heat-killed cells of Bacteroides sp. strain JW20 (a termite gut isolate) for heat-killed rumen bacteria in media as a source of bacterial cell material. Heat-killed Bacteroides sp. strain JW20 was the best of a number of bacteria tested, and under these conditions H₂ was a major protozoan fermentation product. Growth of T. termopsidis strains was further improved by co-cultivation in the presence of Methanospirillum hungatii. M. hungatii was the best of a number of H₂-consuming bacteria tested, and under these conditions CH₄, but not H₂, was produced, indicating interspecies transfer of H₂ between the protozoa and M. hungatii. Both strains of T. termopsidis used powdered, particulate forms of cellulose (e.g., pure cellulose, corncob, cereal leaves) as fermentable energy sources, although powdered wood, chitin, or xylan supported little or no growth. Cells of the cellulose-forming coccus Sarcina ventriculi also served as a fermentable energy source, but these were used poorly as a source of bacterial cell material. The only substantial difference between T. termopsidis 6057 and 6057C was that the latter grew poorly or not at all with rumen bacteria as a source of bacterial cell material. The improved growth of T. termopsidis in vitro should facilitate further studies on the cell biology and biochemistry of these symbiotic, anaerobic protozoa.     

Cellulase and Other Polymer-Hydrolyzing Activities of Trichomitopsis termopsidis, a Symbiotic Protozoan from Termites

Crude extracts of the anaerobic, cellulolytic protozoan Trichomitopsis termopsidis possessed endo-β-1,4-glucanase and cellobiase activities, as evidenced by hydrolytic action on carboxymethyl cellulose and cellobiose, respectively. Cell extracts also hydrolyzed microcrystalline cellulose. Hydrolysis of microcrystalline cellulose displayed optima at pH 5 and at 30°C, and glucose was the sole product liberated. Cellulolytic activities of T. termopsidis appeared to be entirely cell associated. Hydrolytic activity was also detected against Douglas fir wood powder, xylan, starch, and protein, but not chitin. The importance of these enzymes in the nutrition of T. termopsidis is discussed in terms of the natural habitat of this protozoan (the hindgut of wood-eating termites).     

Ethionine and Methionine Metabolism by the Chrysomonad Flagellate Ochromonas danica

SYNOPSIS. Growth of Ochromonas danica is competitively inhibited by ethionine. Inhibition can be reversed by methionine. Inhibition indexes of the effect of ethionine on growth and methionine incorporation into proteins are 1 and 4, respectively. Inside the cell, methionine is partially de-methylated and metabolized to form cysteine. Ethionine is partially de-ethylated, and the homocysteine moiety is either re-methylated to form methionine or further metabolized to form cysteine. Ethionine is also incorporated into proteins of O. danica. The kind of metabolic interference, expressed by inhibition of growth, and correlated with incorporation of ethionine, is yet unknown.     

Ethionine and Methionine Metabolism by the Chrysomonad Flagellate Prymnesium parvum

SYNOPSIS. Ethionine or methionine can serve as sole nitrogen source for growth of Prymnesium parvum. Both amino acids are taken up as such at a ratio of 2 : 1 methionine/ethionine. Ethionine is totally de-ethylated in the cell, while methionine is probably only partially de-methylated. The homocysteine moiety of both amino acids is similarly metabolised to form cysteine or re-methylated to form methionine. De-ethylation of ethionine seems how P. parvum avoids its antimetabolic effect     

Cellulose and Xylan Utilisation in the Lower Termite Reticulitermes speratus

The distribution of the enzymes of cellulose and xylan metabolism namely endo-beta-1,4-glucanase, beta-glucosidase, endo-beta-1,4-xylanase and beta-xylosidase activities, in Reticulitermes speratus (Kolbe) was measured both in the salivary glands and in the major gut sections and along the length of the gut in freshly collected termites. The majority of the endo-beta-1,4-glucanase activity (77.8%) was found in the salivary glands which also contained 23.9% of the beta-glucosidase activity. At least 70% of the remaining activity was located in the anterior section of the hindgut. A small amount of endo-beta-1,4-xylanase activity (2.4%), but no beta-xylosidase activity, was present in the salivary glands. The majority of these activities were in the anterior section of the hindgut. The RQ of freshly collected termites at 25 degrees C was 1.03+/-0.01. Maintaining termites for 16 days on wood, cellulose and xylan showed that the RQ values of termites fed on wood or xylan were not significantly different from those of freshly collected termites but significantly increased when maintained on cellulose. The RQ of starved termites after 11 days was 0.81+/-0.02. There were three effects on protozoan populations of feeding termites xylan for 20 days. One species, Dinenympha parva was not affected, while five others, Pyrsonympha grandis, Holomastigotes elongatum, Dinenympha rugosa, Dinenympha leidy and Dinenympha porteri survived for 20 days but slowly decreased in numbers. The numbers of P. grandis and D. leidy surviving for 20 days were significantly different from those in starved termites. The third group comprising the two large species, Teratonympha mirabilis and Trichonympha agilis and three small species, Pyrsonympha modesta, Dinenympha exilis and Dinenympha nobilis disappeared within 15 days as in starved termites. It is suggested that protozoa in the first two groups are xylanolytic. Protozoan populations on wood and cellulose diets were not markedly affected. Selective removal of the protozoa by u.v. irradiation led to the loss of xylanolytic activity and a life span comparable to starved termites. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Metabolism and Solubilization of Cellulose by Clostridium cellulolyticum H10

When Clostridium cellulolyticum was grown with cellulose MN300 as the substrate, the rates of growth and metabolite production were found to be lower than those observed with soluble sugars as the substrate. At low cellulose concentrations, the growth yields were equal to those obtained with cellobiose. The main fermentation products from cellulose and soluble sugars were the same. Up to 15 mM of consumed hexose, a change in the metabolic pathway favoring lactate production similar to that observed with soluble sugars was found to occur concomitantly with a decrease in molar growth yield. With cellulose concentrations above 5 g/liter, accumulation of soluble sugars occurred once growth had ceased. Glucose accounted for 30% of these sugars. A kinetic analysis of cellulose solubilization revealed that cellulolysis by C. cellulolyticum involved three stages whatever cellulose concentration was used. Analysis of these kinetics showed three consecutive enzymatic activity levels having the same K_m (0.8 g of cellulose per liter, i.e., 5 mM hexose equivalent) but decreasing values of V_max. The hypothesis is suggested that each step corresponds to differences in cellulose structure.     

The Motility Symbiont of the Termite Gut Flagellate Caduceia versatilis Is a Member of the “Synergistes” Group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the “Synergistes” phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The “Synergistes” phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, “Candidatus Tammella caduceiae,” for this unique motility ectosymbiont of Caducaia versatilis.     

The Metabolism of Cellulose-digesting, Symbiotic Flagellates of the Genus Trichonympha from the Termite Zootermopsis

SUMMARY. Manometric and culture experiments were designed to study factors influencing the metabolism of Trichonympha from Zootermopsis. A starvation period of 36–44 hours for the termites lowered the endogenous metabolism of Trichonympha and made possible the testing of substrates in the Warburg apparatus. In the presence of antibiotics, cellulose and cellobiose were utilized at approximately the same rate by the protozoa. Yeast extract stimulated cellulose fermentation in Trichonympha from starved termites, but the effect could not be demonstrated in protozoa from recently fed termites. Yeast extract, blood serum, and liver fractions improved the survival of the flagellates in anaerobic cultures.     

Staining Flagellate Protozoa by Various Silver-Protein Compounds

Seven samples of silver protein (Protargol type) were tested on flagellate protozoa from the alimentary tract of frogs, golden hamsters, and termites. The samples consisted of one of Protargol (Winthrop's prewar German), one of Protargol S (Winthrop-Stearns, Inc., present manufacture, Commission Certified), and five of the experimental batches (4Z, 20, 20B, 22, and 36) by H. A. Davenport and collaborators (1952). The pre-war Protargol is rated best and batch 22 second best for staining of all flagellates. Protargol S gave uniform, but only fair results, with all organisms, while batch 20B, better than Protargol S in several instances, was poorer in a few. Thus Protargol S and batch 20B are rated third, as about equal. Batch 4Z is rated fourth; and batch 20, which stained some species, fifth. Batch 36 stained no protozoa. The tests show that while the present Protargol S is usable for protozoa, it is still inferior to the old German variety. Since some of the experimental batches, none of which was made by exactly the same process, gave promising results, further study of the relation between manufacturing process and subsequent staining reaction should be fruitful.     

Staining Flagellate Protozoa by Various Silver-Protein Compounds

Seven samples of silver protein (Protargol type) were tested on flagellate protozoa from the alimentary tract of frogs, golden hamsters, and termites. The samples consisted of one of Protargol (Winthrop's prewar German), one of Protargol S (Winthrop-Stearns, Inc., present manufacture, Commission Certified), and five of the experimental batches (4Z, 20, 20B, 22, and 36) by H. A. Davenport and collaborators (1952). The pre-war Protargol is rated best and batch 22 second best for staining of all flagellates. Protargol S gave uniform, but only fair results, with all organisms, while batch 20B, better than Protargol S in several instances, was poorer in a few. Thus Protargol S and batch 20B are rated third, as about equal. Batch 4Z is rated fourth; and batch 20, which stained some species, fifth. Batch 36 stained no protozoa. The tests show that while the present Protargol S is usable for protozoa, it is still inferior to the old German variety. Since some of the experimental batches, none of which was made by exactly the same process, gave promising results, further study of the relation between manufacturing process and subsequent staining reaction should be fruitful.     

Intranuclear Parasitism of the Ciliate Euplotes by a Trypanosomatid Flagellate

A trypanosomatid flagellate, Leptomonas sp., develops and multiplies in the macronucleus (only) of natural and laboratory-reared populations of the ciliate Euplotes. Up to 90% of the natural populations of Euplotes in our test pond had such nuclear infections. Laboratory infections were transmitted to this ciliate by feeding it liberated parasites. Paramecium resisted infections. All laboratory-induced infections were lethal to Euplotes, while control clones of the uninfected ciliates remained viable. This leptomonad, unlike Leptomonas karyophilus (found in Paramecium), shows no leishmanial forms in its several ciliate hosts and shows a varied pattern of locomotion.     

Studies on Trichomonads. II. The Metabolism of a Trichomonas batrachorum-type Flagellate from the Cecum of Swine

SYNOPSIS. Certain aspects of the metabolism of a Trichomonas batrachorum -type flagellate from the cecum of swine were studied. This trichomonad (1) oxidized glucose, mannose, maltose, sucrose, and inulin, (2) was incapable of oxidizing Krebs' cycle intermediates, (3) possessed peroxide-splitting capabilities, (4) was inhibited by only iodoacetate and arsenite, and (5) formed acid(s) aerobically. Although there was no effect on oxygen uptake, pyruvate and lactate increased the anaerobic evolution of gas(es). In addition to CO₂, other gas, not absorbed by KOH, was produced anaerobically.
Compared with other porcine trichomonads, the metabolism of this small trichomonad resembles most closely that of the large cecal trichomonad, T. suis. However, the smaller trichomonad had a generally lower respiratory rate, a slightly lower optimal pH, and failed to oxidize fructose, galactose, lactose, raffinose, and trehalose.     

Ultrastructure of the Trichomonad Flagellate Stephanonympha nelumbium

ABSTRACT Stephanonympha nelumbium is a large trichomonad measuring 45–60 μm in length and 20–40 μm in width. It is a member of the multinucleate and multiflagellate family Calonymphidae. While the numerous flagella arise in groups of four at the anterior cell pole, the posterior body portion is covered with attached spirochetes and rod-like bacteria. Generally, in the apical body portion of S. nelumbium , 50–100 nuclei are arranged in five to seven circular rows. Each nucleus is associated with a typical mastigont system, comprised of three anterior flagella, one recurrent flagellum being attached to the cell surface for a certain distance, and several typical root structures. Akaryomastigonts and costas do not occur. The fine structure of S. nelumbium corresponds with that of other calonymphids. The main difference to Calonympha is that the axostyle does not embrace the nucleus but passes it in form of a flattened rod.     

Effect of Temperature and Termite Starvation on Phagocytosis by Protozoan Symbionts of the Eastern Subterranean Termite Reticulitermes flavipes Kollar

Abstract Many termite species rely on intestinal protozoan symbionts to digest their cellulosic foods. We examined cellulose acquisition by the symbionts of the Eastern subterranean termite Reticulitermes flavipes Kollar (Isoptera; Rhinotermitidae) by following their phagocytosis of red paper fed to the termite host. The effects of termite host starvation and environmental temperature on feeding activity were studied in the zooflagellates Trichonympha agilis Leidy (Trichonymphidae), Pyrsonympha vertens Leidy, Dinenympha fimbriata Kirby, and D. gracilis Leidy (Pyrsonymphidae), which are among the largest residents in R. flavipes' hindguts. Protozoans in termites starved for 24 h ingested red paper significantly sooner than protozoans in termites with continuous access to food. Trichonympha, Pyrsonympha, and Dinenympha all ingested red paper particles at approximately the same rate. Red paper appeared significantly sooner in protozoans in termites maintained at 32°C than in those maintained at 22°C or 26°C. At 32°C, numbers of Trichonympha per gut remained constant over 96 h. Pyrsonympha and Dinenympha cells were absent or significantly reduced in number by 72 h at that temperature. These results provide insight into the environmental factors that shape the termite–protozoan symbiosis. They may aid in the development of protozoicides used to control pest termites. Received: 1 August 1997; Accepted: 26 November 1997     

Oxygen and Hydrogen Isotope Fractionation during Cellulose Metabolism in Lemna gibba L

Lemna gibba L. B3 was grown under heterotrophic, photoheterotrophic, and autotrophic conditions in water having a variety of hydrogen and oxygen isotopic compositions. The slopes of the linear regression lines between the isotopic composition of water and leaf cellulose indicated that under the three growth conditions about 40, 70, and 100% of oxygens and carbon-bound hydrogens of cellulose exchanged with those of water prior to cellulose formation. Using the equations of the linear relationships, we estimated the overall fractionation factors between water and the exchanged oxygen and carbon bound-hydrogen of cellulose. At least two very different isotope effects must determine the hydrogen isotopic composition of Lemna cellulose. One reflects the photosynthetic reduction of NADP, while the second reflects exchange reactions that occur subsequent to NADP reduction. Oxygen isotopic composition of cellulose apparently is determined by a single type of exchange reaction with water. Under different growth conditions, variations in metabolic fluxes affect the hydrogen isotopic composition of cellulose by influencing the extent to which the two isotope effects mentioned above are recorded. The oxygen isotopic composition of cellulose is not affected by such changes in growth conditions.     

Culture and Phylogenetic Characterization of Trichomitus trypanoides Duboscq & Grassè 1924, n. comb.: a Trichomonad Flagellate Isolated from the Hindgut of the Termite Reticulitermes santonensis Feytaud

ABSTRACT. A trichomonad flagellate strain R1 was isolated from the hindgut contents of the termite Reticulitermes santonensis Feytaud. The flagellate was cultivated at 28° C in anaerobic medium containing yeast extract, minerals and vitamins. The isolate fed on living bacteria. It showed the typical morphological and ultrastructural features of the trichomonads. closely resembling Trichomitus trypanoides. In order to determine its phylogenetic position the small subunit ribosomal DNA (SSU rDNA) of the flagellate was amplified in vitro using the polymerase chain reaction (PCR), cloned in a plasmid vector and sequenced. Comparison of the obtained sequence with so far available SSU rRNA/rDNA sequences showed strongest similarity (89%) to the sequence of Tritrichomonas foetus. The phylogenetic analysis with parsimony and distance matrix methods placed Trichomitus trypanoides strain R1 near by the root of the phylogenetically so far analyzed eukaryotic organisms. This confirms that termites harbour hindgut symbionts, which originate from very early evolved eukaryotes.