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Marine, sediment-dwelling ciliates were examined for cytochrome oxidase activity by a cytochemical method and for fine structural details. Species of Plagiopylidae (Trichostomatida), i.e. Plagiopyla frontata, Sonderia vorax and Sonderia sp., and of Heterotrichda, i.e., Parablepharisma pellitum, Parablepharisma sp., Metopus contortus, Metopus vestitus and Caenomorpha capucina; previously considered to be obligate anaerobes because of their sulfide-containing habitat, do not have cytochrome oxidase activity or mitochondria with cristae or tubuli. The evolutionary origin and significance of anaerobic ciliates is discussed. Most of the anaerobic ciliates harbor a flora of ecto- and endosymbiotic bacteria as demonstrated by transmission and scanning electron micrographs. It is speculated that the bacteria may utilize the metabolic end products of the protozoa for growth and energy yielding processes. These associations are also compared with other, previously described cases of symbiosis involving prokaryotes and protozoa.
Ciliate encystment is an eukaryotic cell differentiation process which involves a specific gene expression, to form the resting stage. In this study, we investigate, for first time, the DNA methylation pattern changes during encystment in the ciliate Colpoda inflata, and the 5-azacytidine effect on growing cells and encystment. Results indicate that 5-methylcytosine is present in macronuclear DNA of this ciliate and the 5-azacytidine treatment induces encystment in growth conditions. From restriction enzyme digestion and 5-azacytidine experiments, we conclude that a specific DNA demethylation is probably involved in the encystment gene expression of this ciliate.
The effect of the establishment of Entodinium caudatum on the population of Eudiplodinium maggii was examined in the rumen of three sheep fed a hay/ground barley diet. The cell concentration of E. maggii were 15.9-38.5 and 11.7-12.4 x 10(3) cells per g of the rumen contents in the absence and presence of E. caudatum, respectively. Microscopic analysis showed that starch was the only material engulfed by eudiplodinia irrespective of the time after feeding and the presence or absence of E. caudatum. Up to 82-93% of individuals contained starch grains when E. maggii was the only ciliate species in the rumen; the proportion was 70-77% after entodinia had been established. The largest quantity of starch engulfed by E. maggii ciliates was 12.4-19.0 and 6.7-7.6 mg per 100 mg protozoal dry mass in the absence and presence of entodinia, respectively. No visible engulfment of hay was observed in vivo in spite of the fact that hay particles up to 42 microns in length were dominating in rumen fluid. Ingestion of fresh particles of hay separated from the rumen digesta was found when they were added in the proportion of 1 g per 40 mL suspension of ciliates. No preferential intake of starch was observed when E. maggii ciliates were incubated in vitro with a mixture of hay and barley starch. It is suggested that competition for starch between the two ciliate species was responsible for the drop in the numbers of E. maggii. This could result from a too low concentration of small particles of hay in the rumen fluid.
The purpose of this study was to investigate factors affecting mechanical- and cryo-resistance of the rumen ciliates Entodinium caudatum (E.c.), Entodinium furca monolobum (E.f.m.), Entodinium simplex (E.s.), Diplodinium denticulatum (two clones, D.d.01 and D.d.02), Diploplastron affine (D.a.) and Epidinium ecaudatum forma caudatum (E.e.c.) after long-term in vitro cultivation. Following prolonged in vitro cultivation (more than six months), the ciliates were very sensitive to both centrifugation and 5% (v/v) dimethylsulphoxide, with motility decreased to: 39 and 23% for E.c., 66 and 32% for E.f.m., 46 and 27% for D.d. 01, 64 and 41% for D.a., and 44 and 28% for E.e.c., respectively. Thus, cryopreservation was unsuccessful. The effect of supplementing the ciliate growth medium with rumen fluid, glycine-betaine, proline, myo-inositol, linoleic acid, Sel-Plex or insulin, together with the effect of the source of rumen fluid on ciliate resistance to centrifugation, dimethylsulphoxide and freezing was also tested. The omission of rumen fluid from the growth medium resulted in the loss of cryoresistance after one-month cultivation. Supplementing the growth environment with a combination of glycine-betaine, proline, linoleic acid, Sel-Plex, insulin plus improved quality rumen fluid significantly enhanced survival of the ciliates after the freezing-thawing procedure (from 1 to 33% survival in un-supplemented vs. supplemented for E.c., P<0.01; 4-40% E.f.m., P<0.01; 0-17% D.d., P<0.05; 5-7% D.a. and 4-36% E.e.c., P<0.01).
The phylogenetic relationships of members of the ciliate class Litostomatea were determined by a molecular phylogeny using the small subunit of the ribosomal RNA (ssu-rRNA) gene and a morphological phylogeny based on ultrastructural analyses of the group. Molecular analyses consistently supported the monophyly of Trichostomatia, Entodiniomorphida and the "Australian" trichostomes but provided limited support for a monophyletic Vestibuliferida and Haptoria. The results of the morphological analyses depended on the way in which the dataset was treated: "unordered" and "ordered" recovered a monophyletic Trichostomatia, Haptoria and the "Australian" trichostomes but challenged the monophyly of Entodinimorphida and Vestibuliferida; "dollo" recovered a monophyletic Trichostomatia and Entodiniomorphida but at the cost of a greatly longer tree than either "unordered" or "ordered" datasets. The monophyly of each "Australian" trichostome family was supported in all analyses and by both approaches. These results suggest that the trichostome ciliates may have become associated with mammals in Gondwana with the "Australian" trichostome ciliates entering Australia with primitive herbivorous marsupials. Subsequent diversification of the "Australian" families was probably a result of dietary specialization and oral and cortical synapomorphies define each family. We decline at this time to erect a formal taxon name for the "Australian" trichostomes due to the instability of other superfamilial taxa within the Litosomatea and concerns about the stability of tree topology until a better taxon sample of litostome ciliates is available.
A new anaerobic microbial consortium has been discovered: the partners are the ciliated protozoon Trimyema sp. and a single species of methanogen. The consortium has been maintained in culture for more than four years. Each ciliate contains up to 300 symbiotic bacteria; many are relatively small and irregularly disc-shaped, and these are distributed throughout the host's cytoplasm, whereas those which are attached to the ciliate's hydrogenosomes are significantly larger and profusely dentate. This attachment is interpreted as an adaptation to maximize capture by the bacteria of the H2 escaping from hydrogenosomes. The 16S rRNA gene of the symbionts has been partially sequenced, and fluorescent oligonucleotide probes have been constructed and used to detect the different morphotypes of the symbiont within the ciliate. The symbionts belong to a new species of archaeobacterium which is a close relative of the free-living methanogen Methanocorpusculum parvum.
Isotricha jalaludinii n. sp. found in the rumen of lesser mouse deer, Tragulus javanicus, in Malaysia was described and illustrated. This new species is characterized by the location and direction of the vestibulum, shape of the macronucleus, and absence of a dent at the vestibular opening. The presence of single peculiar isotrichid species in the rumen of mouse deer, which is recognized as one of the most primitive ruminants, suggests that the isotrichid ciliates similar to I. jalaludinii and Isotricha intestinalis were established at a fairly early period during the evolution of ruminants.
During a survey of the ciliate protozoal composition of the stomach contents of nine dromedary camels of Egypt, fourteen morphotypes of Entodinium ovumrajae, which has been considered as a species peculiar to camels, were found in six camels. Except for five morphotypes including one originally described as an independent species and its forms, these were newly detected. These morphotypes, divided into three groups, can be identified mainly by the morphology of their ectoplasmic processes. Each camel had on average, about five morphotypes of this species.
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Washed suspensions of the ruminal ciliates, Isotricha prostoma and Entodinium simplex, concentrated C(14)-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Radioautographs demonstrated that oleic acid-1-C(14) was hydrogenated to stearic acid by I. prostoma, and Warburg manometric data showed that the sodium salts of oleic, valeric, caproic, and acetic acids, and methyl myristate, methyl laurate, and the triglyceride tributyrin stimulated fermentation of I. prostoma. The total lipid and free fatty acid contents of I. prostoma were determined.
The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C(14)-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C(14)-labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells.
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The opportunity of the use of formalin-fixed endobiotic ciliates from old collections in transmission electron microscopic investigations (TEM) has been studied. Ciliates from the following species were examined: Ditoxum funinucleum Gassovsky, 1919 from the hindgut of Equus hemionus kulan Groves et Mazak, 1967 preserved in a collection during 19 years, Blepharoprosthium pireum Bundle, 1895 and Cochliatoxum periachtum Gassovsky, 1919 from the hindgut of the Yakut horse Equus caballus L. stored during 1.5 years, and Triplumaria heterofasciculata Timoshenko et Imai, 1995 from faeces of the Asian elephant Elephas maximus L. stored during 2.5 years. It is shown, that the main taxonomically important characters of the cortex ultrastructure, ciliature, and internal fibril structure of the cell of Trichostomatia keep well during a long-term storage in formalin.
Endobiotic ciliates of native Yakut horse are investigated. 57 ciliate species have been found. From 17 up to 43 species of ciliates have been revealed in each host specimen. The specimens of Alloiozona trizona Hsiung, 1930 and Triadinium caudatum Fiorentini, 1890 were present in 100 % of the horses. The endemic genus and species of allantosomides, Strelkowella urunbasiensis Kornilova, 2004, has been found in 11 horses. Allantoxena japonensis (Imai, 1979) and Cycloposthium ishikawai Gassovsky, 1919 being recorded previously in Japan only were found for the first time in Russia. The species Cycloposthium ponomarevi Kornilova, 2001 peculiar to the Turkmenistan koulan has been found in the Yakut horses.