Porphyrin rings and phospholipids: stimulators of cloning efficiency in certain species of Tetrahymena.

Species of Tetrahymena, including T. vorax, T. thermophila, T. pyriformis, and T. pigmentosa, were tested for cloning efficiency in proteose peptone and in synthetic nutrient media to which were added hemin, protoporphyrin IX, chlorophyllin, or asolectin, an impure mixture of phospholipids. All species could be cloned with high efficiency in the crude media. In unsupplemented synthetic medium the cloning efficiencies were 0-10%, around 50%, around 50%, and 90-100% for T. thermophila, T. vorax, T. pyriformis, and T. pigmentosa, respectively. The first three were all stimulated to 90-100% by addition of the porphyrin or phospholipid compounds mentioned above. Uroporphyrin III and coproporphyrin I and III had no effect. We suggest that cells unable to form clones suffer from a lack of cellular energy. This situation may be alleviated by our additions, certain porphyrin rings may be built into cytochromes and phospholipids may be used as fuel. Thus, the synthetic media used so far for these ciliates have not been optimal.     

The 5S and 5.8S ribosomal RNA sequences of Tetrahymena thermophila and T. pyriformis

The nucleotide sequences of the 5S rRNAs of Tetrahymena thermophila and two strains of T. pyriformis have been determined to be identical. The 5.8S rRNA sequences have also been determined; these sequences correct several errors in an earlier report. The 5.8S rRNAs of the two species differ at a single position. The sequencing results indicate that the species are of recent common ancestry. Molecular evidence that has been interpreted in the past as suggestive of an ancient divergence has been reviewed and found to be consistent with a T. pyriformis complex radiation beginning approximately 30-40 million years ago.     

Artabotrys byrsophyllus and A. tipuliferus spp. nov. (Annonaceae) from Peninsular Malaysia and Thailand

Two new species of Artabotrys (Annonaceae) from Peninsular Malaysia and Thailand are described. Conservation assessments for the two new species are made.     

Porphyrin Rings and Phospholipids: Stimulators of Cloning Efficiency in Certain Species of Tetrahymena

ABSTRACT. Species of Tetrahymena , including T. vorax, T. thermophila, T. pyriformis , and T. pigmentosa , were tested for cloning efficiency in proteose peptone and in synthetic nutrient media to which were added hemin, protoporphyrin IX, chlorophyllin, or asolectin, an impure mixture of phospholipids. All species could be cloned with high efficiency in the crude media. In unsupplemented synthetic medium the cloning efficiencies were 0–10%, around 50%, around 50%, and 90–100% for T. thermophila, T. vorax, T. pyriformis , and T. pigmentosa , respectively. The first three were all stimulated to 90–100% by addition of the porphyrin or phospholipid compounds mentioned above. Uroporphyrin III and coproporphyrin I and III had no effect. We suggest that cells unable to form clones suffer from a lack of cellular energy. This situation may be alleviated by our additions: certain porphyrin rings may be built into cytochromes and phospholipids may be used as fuel. Thus, the synthetic media used so far for these ciliates have not been optimal.     

Experimental Infection of the Gray Garden Slug, Deroceras reticulatum (Müller), By the Holotrichous Ciliate Tetrahymena pyriformis (Ehrenberg)

SUMMARY. To determine whether Tetrahymena pyriformis could infect Deroceras reticulatum and become morphologically modified in the same way as the parasitic phase of T. limacis , 50 slugs were exposed for 10 days to an amicronucleate clone of T. Pyriformis and 50 were exposed for the same period of time to a micronucleate clone. The slugs were then transferred to clean containers for another 10 days, to allow ciliates which had entered the slugs either to persist and multiply or, in the case of transitory infections, to disappear. An additional 50 slugs were kept in a control group for 20 days. All of the slugs had been collected at a single locality in which natural infections by T. limacis had not been noted, and were maintained in separate one-half pint milk bottles throughout the experimental period.
Of the slugs exposed to the amicronucleate clone, 40 survived to the end of the experimental period, and 15 were infected. Of those exposed to the micronucleate clone, 42 survived, and 5 were infected. Forty-three of the slugs in the control group survived, and none were infected by ciliates. In some of the slugs infected by T. pyriformis , hundreds of ciliates were noted. The infections were apparently limited to the digestive gland, intestinal tract, and fluid around the viscera. Ciliates recovered from slugs were in general smaller than those growing in thriving cultures, but morphologically were typical T. pyriformis. None were observed to have a proportionately smaller cytostome or apiculate anterior end characteristic of the parasitic phase of T. limacis.     

Two new anamorphic yeasts, Candida thailandica sp. nov. and Candida lignicola sp. nov., isolated from insect frass in Thailand

Two new yeast strains of the genus Candida were isolated from insect frass collected in Khao-Yai National Park, Nakhonrachasima, Thailand. Based on the morphological, physiological and chemotaxonomic characteristics, and sequence analysis of the D1/D2 domain of 26S rRNA gene, these two strains were found to represent two distinct undescribed species and were named Candida thailandica sp. nov. (ST-17 = BCC 7717(T) = NBRC 102562(T)=CBS 10 610) and Candida lignicola sp. nov. (ST-33 = BCC 7733(T) = NBRC 102564(T) = CBS 10612). In the D1/D2 domain of 26S rRNA gene, C. thailandica (GeneBank accession no. AY228491) differs from Candida tsuchiyae, the nearest species, in 66 nucleotide substitutions (10%) and C. lignicola (GeneBank accession no. AY845350) differs from Candida coipomoensis, the nearest species, in nine nucleotides (1.6%). These two new species are clearly distinguished from their closest species by the assimilation of several carbon compounds.     

Lipid modification during cytodifferentiation of Tetrahymena vorax. Whole cell phospholipids and triacylglycerols of microstomal and macrostomal phenotypes

Microstomal cells of the ciliate Tetrahymena vorax V2S can be induced to undergo cytodifferentiation to form an alternate phenotype known as the macrostomal cell; however, sublines of T. vorax exist that respond differently to methods that induce macrostomal cell formation. The phospholipid- and triacylglycerol-bound fatty acid compositions of microstomal and macrostomal cells of a high-transforming subline (designated 3-C) were determined and compared to similar data from cells of a low-transforming subline (designated Ala). Differences in fatty acid composition were found between the two phenotypes as well as between the different sublines. Some change in the distribution of radioactive acetate and lauric acid into phospholipid classes of the different subline was observed, and evidence was also obtained that indicated changes in the relative amounts of the sterol-like pentacyclic triterpenoid tetrahymanol. A limited analysis of the lipid composition of stomatin revealed the presence of small amounts of tetrahymanol, phospholipid and free fatty acid. Stomatin is the naturally produced material obtained from T. pyriformis that triggers differentiation in T. vorax. The existence of a low-transforming subline provides a powerful experimental tool for elucidating the underlying biochemical and molecular mechanisms that control cytodifferentiation in T. vorax and possibly in other eukaryotic cells.     

Isozymic Characterization of Three Mating Groups of the Tetrahymena pyriformis complex*

SYNOPSIS. Strains of 3 unnamed mating groups of the Tetrahymena pyriformis complex have been subjected to starch gel electrophoresis followed by staining the gels for the enzymes isocitrate dehydrogenase (NADP), tyrosine aminotransferase, and tetrazolium oxidase (superoxide dismutase). With respect to the electrophoretic mobilities of these enzyme systems, the mating groups referred to here as 5, 13 and 14 are very similar to Tetrahymena americanis (syngen 2), the most common North American species of the complex. Cultures in our collection labeled Tetrahymena cosmopolitans (formerly syngen 4) are either amicronucleate, with unique isozyme patterns, or micronucleate cells which mate with and have isozyme patterns similar to Tetrahymena canadensis (syngen 7). Immature progeny have been derived from crosses between the latter strains and T. canadensis recently collected in Colorado. The amicronucleate strains are now placed in the Tetrahymena sp. category, and we conclude that strains identifiable as T. cosmopolitanis are no longer available. The reliability of isozymes as characters in ciliate taxonomy was evaluated by comparing the present results for 3 enzymes in 15 groups of strains (syngens and phenosets) that had been compared in an earlier study. These enzyme systems gave correlation coefficients (r) of 0.75 or higher in the separate studies, and can be considered useful diagnostic traits. Other enzymes that were present at threshold levels of detectability or varied highly in concentration from species to species are too unreliable to be of diagnostic value. Some of the strains in the complex are so evolutionarily divergent at the molecular level that we have difficulty finding growth and electrophoretic conditions under which orthologous enzyme activities can be detected simultaneously for all the strains being compared.     

Hoya somadeeae sp. nov. (Apocynaceae,Asclepiadoideae) Thailand and lectotypification of Hoya wrayi

A new species from evergreen montane forests of Thailand, Hoya somadeeae Rodda & Simonsson is here described and illustrated. The new species may be superficially confused with the Peninsular Malaysia endemic H. wrayi King & Gamble, but is clearly separated from it by the very elongated peduncles, up to 20 cm long, the revolute corollas, and outer processes of corona lobes with a membranaceous basal appendage. Morphological affinities between the new species and other Hoya species from Thailand and neighbouring Myanmar and Peninsular Malaysia are discussed.     

New Paramecium quadecaurelia strains (P. aurelia spp. complex,Ciliophora) identified by molecular markers (rDNA and mtDNA)

Paramecium quadecaurelia is a rare species (previously known only from two locations) belonging to the P. aurelia species complex. In the present paper, fragments of an rDNA gene (ITS1-5.8S-ITS2-5′ rDNA) and mtDNA genes (cytochrome oxidase subunit I and cytochrome b regions) were employed to assist in the identification and characterization of three new strains collected from Ecuador and Thailand. Molecular data were confirmed by mating reactions. In rDNA and mtDNA trees constructed for species of the P. aurelia complex, all P. quadecaurelia strains, including the three new strains discussed in this study and two known previously from Australia and Africa, form a monophyletic but differentiated clade. The present study shows that genetic differentiation among the strains of P. quadecaurelia is equal to or even greater than the distances between some other P. aurelia species, e.g., P. primaurelia and P. pentaurelia. Such great intra-specific differentiation may indicate a future splitting of the P. quadecaurelia species into reproductively isolated lines.     

东方人类血吸虫及其螺蛳宿主交互感染的研究

以我国安徽及菲律宾莱特岛的日本血吸虫、湄公血吸虫、马来西亚类似日本血吸虫以及中华血吸虫交互感染其螺蛳宿主。结果表明日本血吸虫及钉螺与湄公血吸虫、马来西亚类似日本血吸虫及其螺蛳宿主亲缘关系较疏远。而湄公血吸虫与马来西亚类似日本血吸虫及其螺蛳宿主有较近的亲缘关系。交互感染结果为马来西亚类似日本血吸虫及钙河罗氏螺的分类问题提供了新的依据。     

Conserved cis- and trans-acting determinants for replication initiation and regulation of replication fork movement in tetrahymenid species.

The rDNA minichromosomes of Tetrahymena thermophila and Tetrahymena pyriformis share a high degree of sequence similarity and structural organization. The T.thermophila 5' non-transcribed spacer (5' NTS) is sufficient for replication and contains three repeated sequence elements that are conserved in T.pyriformis , including type I elements, the only known determinant for replication control. To assess the role of conserved sequences in replication control, structural and functional studies were performed on T.pyriformis rDNA. Similar to T.thermophila , replication initiates exclusively in the 5' NTS, localizing to a 900 bp segment. Elongating replication forks arrest transiently at one site which bears strong similarity to a tripartite sequence element present at fork arrest sites in T.thermophila rDNA. An in vitro type I element binding activity indistinguishable from the T.thermophila protein, ssA-TIBF, was detected in T.pyriformis extracts. The respective TIBF proteins bind with comparable affinity to type I elements from both species, suggesting that in vivo recognition could cross species boundaries. Despite these similarities, the T.pyriformis 5' NTS failed to support replication in transformed T.thermophila cells, suggesting a more complex genetic organization than previously realized.     

Candida kazuoi sp. nov. and Candida hasegawae sp. nov., two new species of ascomycetous anamorphic yeasts isolated from insect frass in Thailand

Two strains of anamorphic yeasts isolated from insect frass collected in southern Thailand were assigned to the genus Candida based on the conventional taxonomic criteria used for yeast classification. In the phylogenetic tree based on the D1/D2 domain of the 26S rDNA, these strains are distant from the known species of yeasts and considered to represent two different new species. They are named Candida kazuoi sp. nov. and Candida hasegawae sp. nov.     

Genetic diversity of taro, Colocasia esculenta (L.) Schott, in Southeast Asia and the Pacific

The genetic diversity of 255 taro (Colocasia esculenta) accessions from Vietnam, Thailand, Malaysia, Indonesia, the Philippines, Papua New Guinea and Vanuatu was studied using AFLPs. Three AFLP primer combinations generated a total of 465 scorable amplification products. The 255 accessions were grouped according to their country of origin, to their ploidy level (diploid or triploid) and to their habitat—cultivated or wild. Gene diversity within these groups and the genetic distance between these groups were computed. Dendrograms were constructed using UPGMA cluster analysis. In each country, the gene diversity within the groups of wild genotypes was the highest compared to the diploid and triploid cultivars groups. The highest gene diversity was observed for the wild group from Thailand (0.19), the lowest for the diploid cultivars group from Thailand (0.007). In Malaysia there was hardly any difference between the gene diversity of the cultivars and wild groups, 0.07 and 0.08, respectively. The genetic distances between the diploid cultivars groups ranges from 0.02 to 0.10, with the distance between the diploid accessions from Thailand and Malaysia being the highest. The genetic distances between the wild groups range from 0.05 to 0.07. First, a dendrogram was constructed with only the diploids cultivars from all countries. The accessions formed clusters largely according to the country from which they originated. Two major groups of clusters were revealed, one group assembling accessions from Asian countries and the other assembling accessions from the Pacific. Surprisingly, the group of diploid cultivars from Thailand clustered among the Pacific countries. Secondly, a dendrogram was constructed with diploid cultivated, triploid cultivated and wild accessions. Again the division of the accessions into an Asian and a Pacific gene pool is obvious. The presence of two gene pools for cultivated diploid taro has major implications for the breeding and conservation of germplasm.     

Proteolytic degradation of ribosomal proteins during isolation of ribosomes and ribosomal subunits from Tetrahymena

A preparation procedure previously used to isolate active ribosomal subunits from an amicronucleate strain of Tetrahymena of undefined phenoset (T. «pyriformis CGL) yields inactive subunits when applied to other amicronucleate or to micronucleate strains of this protozoa.Proteolytic degradation of a small number of ribosomal proteins during preparation of ribosomal subunits from these strains explains this results. If cell extraction and ribosome isolation are carried out in the presence of iodoacetamide, proteolytic activity is inhibited and active ribosomal subunits are obtained. Comparison of the protein complements of active ribosomal subunits prepared in the presence of iodoacetamide from three amicronucleate strains of Tetrahymena reveals small but significant differences.     

Induced Resistance to 6-Methylpurine and Cycloheximide in Tetrahymena. II. Variation within Vegetative Cultures of Micronucleate T. THERMOPHILA and Amicronucleate T. PYRIFORMIS

Resistance to 6-methylpurine and cycloheximide has been induced in both the micronucleate species Tetrahymena thermophila and the amicronucleate species T. pyriformis. Resistance follows only after mutagen treatment and vegetative growth. The frequencies with which resistant variants are induced and the independence of mutagenesis and selection are demonstrated. All evidence is consistent with the hypothesis that macronuclear subunits are assorting in both species during vegetative growth to produce new phenotypes among subclones.     

Three new thermotolerant methylotrophic yeasts, Candida krabiensis sp. nov., Candida sithepensis sp. nov., and Pichia siamensis sp. nov., isolated in Thailand

Five strains of thermotolerant methylotrophic yeasts isolated in Thailand were found to represent three new species in the genera Pichia and Candida, based on phylogenetic analysis of D1/D2 domain of 26S rDNA, in addition to the morphological, physiological, biochemical and chemotaxonomic characterization. Three strains, FS96 and FS101 from flowers and M02 from tree flux, were characterized by ubiquinone Q7, multilateral budding, and the formation of hat-shaped ascospores that are liberated at maturation. These strains showed identical nucleotide sequences in the D1/D2 domain and formed a cluster with Candida thermophila, "Pichia salicis" and Pichia angusta. They differed by 1.9% of nucleotide substitutions from Candida thermophila, the nearest species. They were considered to represent a single new species and are described as Pichia siamensis sp. nov. Two strains, N051 and S023, isolated from soil did not produce ascospores, proliferated by multilateral budding, did not demonstrate urease or DBB color reaction, and lacked sexual stages. These characteristics correspond to the genus Candida. Strains N051 and S023 differed by 2.8% and 1.9% of nucleotide substitutions in the D1/D2 domain from the nearest species, Candida nemodendra and Candida ovalis, respectively, and are considered to represent respective new species. N051 and S023 are described as Candida krabiensis sp. nov. and Candida sithepensis sp. nov., respectively.     

Two new species of Tubificoides (Oligochaeta, Tubificidae) and new records of T. brownae and T. imajimai from the Gulf of Mexico and Caribbean, with a redescription of T. bakeri

Two new species of Tubificoides are described from the west coast of Florida. Both species have large lateral openings of the penis sheaths with proximal flap-like projections. Tubificoides pollex sp.n. is characterized by (1) papillate body wall in postclitellar segments; (2) possession of hair and bifid setae anteriorly, and only bifid setae in pcstclitellar segments; and (3) conical penis sheaths with a lateral opening and a flap-like projection on the proximal margin. The setal distribution of the new species is similar to that of T. bakeri Brinkhurst, 1986, which is redescribed on the basis of new material. Tubificoides lunatus sp.n. is similar to T. pollex except that the flap-like projections of the penis sheaths are crescent shaped, all the somatic setae are bifid, and body wall papillae are absent. Records of Tubificoides brownae Brinkhurst & Baker, 1979, and T. imajimai Brinkhurst, 1986, from the Gulf of Mexico and Caribbean, respectively, are also presented. The latter species was previously known only from subtropical regions of Japan and the People's Republic of China.     

Systematic review of the Mastacembelidae or spiny eels of Burma and Thailand,with description of two new species ofMacrognathus

Burma and Thailand are inhabited by 14 species of Mastacembelidae, eightMacrognathus and sixMastacembelus. Two new species ofMacrognathus are described from Thailand, one with rostral toothplates and one without.Mastacembelus dayi, known only from Burma, is a valid species related toM. alboguttatus;Mastacembelus favus, from Thailand and Western Malaysia, is distinct from its close relativeM. armatus.