Order Entodiniomorphida Research Articles

Improved and easy method for long-term in vitro culture of rumen ciliates Dasytricha ruminantium

The rumen ciliates are a diverse group of protozoa residing in the rumen of ruminant animals. They are primarily found in the orders Entodiniomorphida and Vestibuliferida, playing crucial roles in the digestion and breakdown of feed within the host's rumen, closely intertwined with the host's nutrient absorption. In vitro monocultures of representatives of rumen ciliates are important to better study them. So far, Entodinium caudatum and Epidinium caudatum, representatives of the order Entodiniomorphida, have been successfully cultivated as a monoculture in vitro. However, for the order Vestibuliferida, no representative species has been established a stable monoculture in vitro up to date, which hampers to study their physiology and metabolism. Therefore, we have developed a simple method for the in vitro cultivation of Dasytricha ruminantium, a representative rumen ciliate in the order Vestibuliferida. Utilizing an optimized culture medium with easily obtainable components, and the cultivation process is simple. This will facilitate further research in metabolism and other studies requiring large pure live materials.1.Filtration and separation for enriching D. ruminantium.2.A culture medium (DRM) suitable for the growth of D. ruminantium, with easily obtainable components.3.Simple cultivation process, facilitating the obtainment of a large number of monocultured D. ruminantium.

Bozasella gracilis n. sp. (Ciliophora, Entodiniomorphida) from Asian elephant and phylogenetic analysis of entodiniomorphids and vestibuliferids

Bozasella gracilis n. sp. in the order Entodiniomorphida was found in fecal samples of an Asian elephant kept in a zoo. The ciliate has general and infraciliary similarities to the families Ophryoscolecidae and Cycloposthiidae. Phylogenetic trees were inferred from 18S rRNA gene sequences of B. gracilis, 45 entodiniomorphids, 10 vestibuliferids, 5 macropodiniids, and an outgroup, using maximum likelihood, Bayesian inference, and neighbor joining analyses. Of them, there were 32 new sequences; 26 entodiniomorphid species in the genera, Bozasella, Triplumaria, Gassovskiella, Ditoxum, Spirodinium, Triadinium, Tetratoxum, Pseudoentodinium, Ochoterenaia, Circodinium, Blepharocorys, Sulcoarcus, Didesmis, Alloiozona, Blepharoconus, Hemiprorodon, and Prorodonopsis, and 6 vestibuliferid species in the genera, Buxtonella, Balantidium, Helicozoster, Latteuria, and Paraisotricha. Thirty additional sequences were retrieved from the GenBank database. Phylogenetic trees revealed non-monophylies of the orders Entodiniomorphida and Vestibuliferida, the suborders Entodiniomorphina and Blepharocorythina, and the families Cycloposthiidae and Paraisotrichidae. Bozasella gracilis was sister to Triplumaria. In addition, to avoid homonymy, we propose Gilchristinidae nom. nov., Gilchristina nom. nov. and Gilchristina artemis (Ito, Van Hoven, Miyazaki & Imai, 2006) comb. nov.

Infraciliature of eight Triplumaria species (Ciliophora, Entodiniomorphida) from Asian elephants with the description of six new species

Intestinal ciliates excreted in the feces of Asian elephants were surveyed. Fourteen species in the order Entodiniomorphida were detected. Nine Triplumaria species in the family Cycloposthiidae were found. Using the silver impregnation, two known species, T. antis and T. dvoinosi, were redescribed and six new species, T. sukuna n. sp., T. zuze n. sp., T. solea n. sp., T. suwako n. sp., T. fulgora n. sp., and T. harpagonis n. sp., were described. T. sukuna, T. zuze, T. solea, and T. suwako have the perivestibular polybrachykinety along the vestibular opening. The buccal infraciliary bands of T. suwako are similar to those of T. selenica found from elephants and the buccal infraciliary bands of T. sukuna, T. zuze, and T. solea are similar to those of T. grypoclunis described from rhinoceroses. T. antis, T. dvoinosi, T. fulgora, and T. harpagonis have the vestibular polybrachykinety extending down inside the vestibulum as found in rumen ciliates in the family Ophryoscolecidae. The caudalial ciliary zones of T. dvoinosi and T. fulgora were retractable as found in rumen ophryoscolecids. Raabena bella and Pseudoentodinium elephantis showed high composition values over 30%. Ciliate densities in the three fecal samples were 0.15, 1.09, and 2.07×104/ml.

Two new entodiniomorphid Triplumaria ciliates from the intestine of the wild African white rhinoceros

Two new species of Triplumaria in the order Entodiniomorphida, T. alluvia n. sp. and T. grypoclunis n. sp., are described from the large intestine of the wild African white rhinoceros. T. alluvia has three bud-shaped caudalia, one broad skeletal plate with a wavy left dorsal edge, and an axe-shaped tail flap. T. grypoclunis has three short arched caudalia, two broad skeletal plates, and a pointed and ventrally curved tail flap. These two new species have a C-shaped adoral polybrachykinety, a slender perivestibular polybrachykinety, and paralabial kineties in their retractable adoral ciliary zone. In T. alluvia, the perivestibular polybrachykinety is joined to both ends of the adoral polybrachykinety and paralabial kineties along the ventral side of the adoral polybrachykinety, showing the same arrangement as in Cycloposthium species. In T. grypoclunis, the perivestibular polybrachykinety is joined only to the right end of the adoral polybrachykinety and paralabial kineties along the left ventral side of the adoral polybrachykinety, showing an arrangement analogous to the Tripalmaria species.

New entodiniomorphid ciliates from the intestine of the wild African white rhinoceros belong to a new family, the Gilchristidae

Gilchristia artemis n.g., n.sp. and Digilchristia draconis n.g., n.sp. in the order Entodiniomorphida are described from the large intestine of the African white rhinoceros, and a new family Gilchristidae is proposed to contain them. These new species have a C-shaped adoral polybrachykinety, a slender vestibular polybrachykinety, and paralabial kineties along the ventral side of the adoral polybrachykinety in their retractable adoral ciliary zone, showing the same arrangement as in the rumen ciliates in the family Ophryoscolecidae. G. artemis has two skeletal plates and D. draconis one plate. In both species the dorsal skeletal plate is bow-shaped, folded in half longitudinally, twisting in the anterior part, and lying along the dorsal left side of the macronucleus. The second plate of G. artemis is slender and lies along the ventral side of the macronucleus. G. artemis has three ciliary arches and D. draconis has four arches along the dorsal and ventral sides of the body. Their arches are long and non-retractable, closely resembling those of ciliates in the families, Spirodiniidae and Cycloposthiidae, and are not analogous to the single retractable ciliary arch of the rumen ciliates in the family Ophryoscolecidae.

Molecular Phylogeny of Litostome Ciliates (Ciliophora, Litostomatea) with Emphasis on Free-Living Haptorian Genera

The monophyly of the litostomes was tested using nine newly sequenced and four previously unpublished small subunit ribosomal RNA (SSrRNA) gene sequences from free-living Haptoria as well as from endosymbiotic Trichostomatia: the vestibuliferids Balantidium coli and Isotricha prostoma, the cyclotrichiid Mesodinium pulex, and the haptorids Loxophyllum rostratum, Dileptus sp., Enchelyodon sp., Enchelys polynucleata, Epispathidium papilliferum (isolates A and B), Spathidium stammeri, Arcuospathidium muscorum, Arcuospathidium cultriforme, and the unusual Teuthophrys trisulca. Phylogenetic analyses depicted the litostomes as a monophyletic group consisting of the trichostomes (subclass Trichostomatia) and the free-living haptorians (subclass Haptoria). The cyclotrichiids Mesodinium and Myrionecta (order Cyclotrichiida) branched either basally within or outside the Litostomatea. In most analyses, the haptorians did not receive support as a monophyletic group. Instead, Dileptus branched basally to all litostome taxa, and Epispathidium papilliferum grouped with the Subclass Trichostomatia. Some subgroupings, however, of haptorian genera corresponded to suggested superfamilial taxa (e.g., orders Spathidiida and Pleurostomatida). Within the monophyletic trichostomes, we can distinguish three clades: (1) an Australian clade; (2) the order Entodiniomorphida; and (3) the order Vestibuliferida. However, Balantidium, currently classified in the Vestibuliferida, did not group with the other vestibuliferids, suggesting that this order may be paraphyletic.

Horizontal gene transfer from Bacteria to rumen Ciliates indicates adaptation to their anaerobic, carbohydrates-rich environment

BackgroundThe horizontal transfer of expressed genes from Bacteria into Ciliates which live in close contact with each other in the rumen (the foregut of ruminants) was studied using ciliate Expressed Sequence Tags (ESTs). More than 4000 ESTs were sequenced from representatives of the two major groups of rumen Cilates: the order Entodiniomorphida (Entodinium simplex, Entodinium caudatum, Eudiplodinium maggii, Metadinium medium, Diploplastron affine, Polyplastron multivesiculatum and Epidinium ecaudatum) and the order Vestibuliferida, previously called Holotricha (Isotricha prostoma, Isotricha intestinalis and Dasytricha ruminantium).ResultsA comparison of the sequences with the completely sequenced genomes of Eukaryotes and Prokaryotes, followed by large-scale construction and analysis of phylogenies, identified 148 ciliate genes that specifically cluster with genes from the Bacteria and Archaea. The phylogenetic clustering with bacterial genes, coupled with the absence of close relatives of these genes in the Ciliate Tetrahymena thermophila, indicates that they have been acquired via Horizontal Gene Transfer (HGT) after the colonization of the gut by the rumen Ciliates.ConclusionAmong the HGT candidates, we found an over-representation (>75%) of genes involved in metabolism, specifically in the catabolism of complex carbohydrates, a rich food source in the rumen. We propose that the acquisition of these genes has greatly facilitated the Ciliates' colonization of the rumen providing evidence for the role of HGT in the adaptation to new niches.

Identification of rumen ciliates using small subunit ribosomal RNA (18S‐rRNA)‐targeted oligonucleotide probes and fluorescence in situ hybridization (FISH)

Up to half of the biomass in the rumen can be represented by ciliates, which play an important role in the digestion processes of their hosts. In the literature, very little information can be found on determination of the diversity of complex rumen ciliate communities. The identification of these fast moving protists is mainly based on live observations and comparisons of their highly variable cell morphologies. It makes accurate identification and quantification of rumen ciliates very difficult if not impossible. The development of fluorescence in situ hybridization (FISH), established as a technique to identify prokaryotes and eukaryotes using ribosomal RNA‐targeted fluorescently labeled oligonucleotide probes, is a promising approach to identify and investigate rumen ciliates. The present study, part of CIMES (ciliates as monitors for environmental safety), a project sponsored by the European Commission, shows the problems of applying FISH on rumen ciliates and how to solve them. Tests resulted in a new protocol, which recommends para‐formaldehyde and formaldehyde at 1–2% final concentration to preserve the ciliates before applying FISH. Furthermore, seven new oligonucleotide probes could be developed and successfully be tested to identify different rumen ciliate taxa of the order Entodiniomorphida (class Litostomatea) by applying FISH. It is also shown, how FISH together with confocal laser scanning microscopy can improve analyses of ciliate cell morphologies. Thanks to Peter Pristas, Peter Javorsky, Ralf Einspanier, Susanne Ulbrich (providing rumen samples), Seung Yeo Moon‐van der Staay, Georg van der Staay (providing unpublished 18S‐rDNA sequences), the European Commission (financial support, project QLK3‐CT‐2002‐02151).

Monophyly of the Trichostome ciliates (Phylum ciliophora: Class litostomatea) tested using new 18S rRNA sequences from the Vestibuliferids, Isotricha intestinalis and Dasytricha ruminantium, and the Haptorian, Didinium nasutum

Summary The monophyly of the trichostomes was tested using new 18S rRNA gene sequences from the rumen ciliates, Dasytricha ruminantium (1,638 bp) and Isotricha intestinalis (1,639 bp), and the haptorian, Didinium nasutum (1,643 bp). In all parsimony and distance trees, Dasytricha and Isotricha always paired together as the vestibuliferids (Order Vestibuliferida). The vestibuliferids were united with a clade consisting of the entodiniomorphid rumen ciliates (Order Entodiniomorphida) to form the trichostomes (Subclass Trichostomatia). In the alternative maximum parsimony tree and distance-matrix trees, Didinium grouped with Homalozoon and Spathidium to form a clade (Order Haptorida) that was the sister group to the pleurostome, Loxophyllum (Order Pleurostomatida). Together, these latter four genera represent haptorian ciliates (Subclass Haptoria), which along with their sister group, the trichostomes, comprise the litostomes (Class Litostomatea).

A new family of entodiniomorph protozoa from the marsupial forestomach, with descriptions of a new genus and five new species.

A unique group of entodiniomorph protozoa was found in forestomach contents from quokka (Setonix brachyurus), western grey kangaroo (Macropus fuliginosus), red kangaroo (Macropus rufus) and euro (Macropus robustus erubescens). A new genus, Macropodinium n.g., containing five new species, is described. Three species are described from forestomach contents of the quokka: Macropodinium baldense n. sp., Macropodinium moiri n. sp. and Macropodinium setonixum n. sp. A single species, Macropodinium ennuensis n. sp., is described from the red kangaroo and euro. The last species, Macropodinium yalanbense n. sp., is described in forestomach contents from the western grey kangaroo. At least three distinct features in the new genus are incompatible with any of the described families in the order Entodiniomorphida. On this basis, the new family Macropodiniidae has been created.

A new family, genus, and seven new species of Entodiniomorphida (Protozoa) from the gut of African rhinoceros.

This report deals with a group of ciliated protozoa with short ciliary bands found mainly in the cecum of black rhinoceros, Diceros bicornis (linnaeus, 1758), and white rhinoceros, Ceratotherium simum (Burchell, 1817) from southern Africa. A new genus, Rhinozeta, based on the sum total of the characteristics of seven new related species is described. The species described are R. rhinozeta n. sp., R. triciliata n. sp., R. caecalis n. sp., R. addoensis n. sp., R. cristata n. sp., R. multiplatus n. sp., and R. unilaminatus n. sp. The specific features of the new genus make it incompatible with any of the known families of the Order Entodiniomorphida containing the ciliates present in the digestive tract of herbivorous mammals. This merits the creation of a new family, the Rhinozetidae.

The Functional and Evolutionary Significance of the Ultrastructure of the Ophryoscolecidae (Order Entodiniomorphida)1

ABSTRACT. Ophryoscolecid ciliates from the order Entodiniomorphida show a series of morphological types which have been interpreted as an evolutionary lineage. In this study, ultrastructural information from three species—the ancestral Entodinium, intermediate Eudiplodinium maggii, and the advanced Epidinium—has been evaluated in terms of ophryoscolecid evolution. The infraciliature, nuclei, contractile vacuoles, cortex, and cytoplasm are all very similar and sometimes indistinguishable among the three species, suggesting a close relationship. The cytoalimentary system, however, shows considerable interspecific variation. The cytopharynx differs in position and extent of microtubules and microfilaments, varying in appearance from a microtubular to a microfilament‐based mechanism while retaining similar component structures. The esophagus, a zone of cytoplasm extending from the cytopharynx and delimited by a microtubular/fibrillar wall, is rudimentary in Entodinium, sac‐like in Eudiplodinium, and tube‐like in Epidinium, where it also has convoluted walls and a sheath of fibrous material that suggest an expansible‐contractile structure. These variations have been related to type of food particle ingested. The capability of the cytoalimentary systems seems to be increased so that the more advanced forms can exploit a food resource, in the form of large plant fragments in the ruminal fluid, not available to the simpler, ancestral forms, which tend to ingest small particles such as bacteria and starch grains. The original evolutionary lineage based on morphological studies using light microscopy is supported by our observations, in these three forms, of ultrastructural variations in the cytopharynx and in their relationship with diet via possible ingestion mechanisms. Additional support for this evolutionary analysis comes from preliminary studies of other ophryoscolecids in which the cytoalimentary organization is consistent with their positions relative to one another in the evolutionary scheme.

Association of methanogenic bacteria with rumen ciliates.

In 11 species of rumen ciliates belonging to nine genera of the family Ophryoscolecidae (order Entodiniomorphida) an ectosymbiosis with methanogenic bacteria was found. The bacteria could be identified as methanogens on the basis of the presence of specific fluorescent coenzymes (F350 and F420). This somatic interaction may reflect a metabolic interaction in which efficient interspecies hydrogen transfer benefits both partners.

Remarks on the Composition of the Large Ciliate Class Kinetofragmophora de Puytorac et al., 1974, and Recognition of Several New Taxa Therein, with Emphasis on the Primitive Order Primociliatida N. Ord.*

SYNOPSISWith the realization that new data (especially ultrastructural) and new ideas are making necessary a major revision of the scheme of classification of the Ciliophora, several groups of ciliatologists are preparing treatises on the subject. The present paper is concerned with the composition of the large new class of ciliates, Kinetofragmophora de Puytorac et al., 1974, established very recently by the French group. Several new taxa, at ordinal and subordinal levels, are proposed for inclusion in that class, with special emphasis on the new order to contain the most primitive of extant species. Actions taken here are incorporated in a major review and revisory work of the author which is being published elsewhere.The class Kinetofragmophora, by far the largest of the 3 classes now recognized as comprising the whole phylum Ciliophora, is itself considered to contain 4 sizeable subclasses and to embrace a total of 13 orders and 14 suborders. Two orders and 6 suborders are named and described here as new, enumerated and briefly identified as follows: Order Primociliatida n. ord., for the most “primitive” of gymnostomes, with three new suborders—Homokaryotina n. subord., for the homokaryotic genus Stephanopogon; Karyorelictina n. subord., for a number of mostly interstitial ciliates which, though heterokaryotic, possess nondividing, diploid macronuclei (e.g. Trachelocerca, Trachelonema, and Tracheloraphis); and Prorodontina n. subord., for a group of relatively specialized formerly “rhabdophorine” gymnostomes such as Coleps, Placus, and Prorodon and order Haptorida n. ord., for rapacious carnivorous forms, formerly lumped with the preceding groups as “rhabdophorines,” many with oral toxicysts and well developed thigmotactic ciliature (e.g. Actinobolina, Didinium, Dileptus, Enchelys, Spathidium, and Trachelius). All foregoing taxa are members of the 1st kinetofragmophoran subclass, the Gymnostomata. In the taxonomic conclusions drawn, new significance is placed on ultrastructural data, on macronuclear differences of evolutionary importance, and on habitat and behavior. A brief review of the literature on psammophilous ciliates is presented.In the subclass Vestibulifera is now located the order Entodiniomorphida Reichenow, a group formerly considered to be a spirotrich taxon. A suborder, Blepharocorythina n. subord., is proposed to contain the old “trichostome” family Blepharocorythidae, species commensalistic in horses and ruminants and now—with their syncilia, etc.—considered ancestral to the ophryoscolecids and relatives.In the subclass Hypostomata, order Nassulida, the suborder Paranassulina n. subord. is established to contain nassulids which appear more highly evolved than Nassula itself (e.g. Paranassula and Enneameron) in perioral ciliature, mode of stomatogenesis, etc. In the enigmatic and still vexatious order Rhynchodida, the suborder Aneistrocomina n. subord. is erected to embrace rhynchodid genera with an anteriorly located sucking tentacle (and other unique characteristics)—for example, Ancistrocoma, Crebricoma, Holocoma, and Sphenophrya. With the banishment of the bulk of the old “thigmotrichs” to the oligohymenophoran order Scuticociliatida, the ancistrocomines are left with the family Hypocomidae (and relatives) in the order Rhynchodida. It is not yet clear, however, how closely related the 2 suborders of rhynchodids should be considered. Special nomenclatural problems are also involved.