Terue Harumoto

Chemical defense by means of pigmented extrusomes in the ciliate Blepharisma japonicum

Summary The defensive function of pigment granules in Blepharisma japonicum against the predatory ciliate Dileptus margaritifer was further investigated by 1) observing the discharge of pigment granules of Blepharisma as a response to the attack by the predator and 2) measuring the toxicity of purified blepharismin, the red pigment localized in pigment granules. When a Blepharisma was attacked by the toxicysts-bearing proboscis of a Dileptus , the Blepharisma instantly released a mass of reddish material at the attacked site. The Dileptus retreated and the Blepharisma swam away. The observation suggested that the Blepharisma discharged pigment granules as a response to the attack. This assumption was confirmed by scanning-electronmicroscopic observations; many pigment granules near the attacked site were discharged at the moment of the attack. Purified blepharismin was highly toxic to Dileptus and several other ciliates, but was not toxic to Blepharisma . We also showed that blepharismin is toxic in the dark. These results strongly support the previously presented hypothesis that the defensive function of pigment granules in B. japonicum against D. margaritifer is based on the discharge of blepharismin as a response to the attack by the predator. We conclude that pigment granules of B. japonicum are extrusomes (extrusive organelles in protists) for chemical defense.

Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum

Conjugation in Blepharisma japonicum is induced by interaction between complementary mating-types I and II, which excrete blepharmone (gamone 1) and blepharismone (gamone 2), respectively. Gamone 1 transforms type II cells such that they can unite, and gamone 2 similarly transforms type I cells. Moreover, each gamone promotes the production of the other gamone. Gamone 2 has been identified as calcium-3-(2′-formylamino-5′-hydroxy-benzoyl) lactate and has been synthesized chemically. Gamone 1 was isolated and characterized as a glycoprotein of 20–30 kDa containing 175 amino acids and 6 sugars. However, the amino acid sequence and arrangement of sugars in this gamone are still unknown. To determine partial amino acid sequences of gamone 1, we established a method of isolation based on the finding that this glycoprotein can be concentrated by a Con A affinity column. Gamone 1 is extremely unstable and loses its biological activity once adsorbed to any of the columns that we tested. By using a Con A affinity column and native PAGE, we detected a 30-kDa protein corresponding to gamone 1 activity and determined the partial amino acid sequences of the four peptides. To isolate gamone 1 cDNA, we isolated mRNA from mating-type I cells stimulated by synthetic gamone 2 and then performed rapid amplification of cDNA ends procedures by using gene-specific primers and cloned cDNA of gamone 1. The cDNA sequence contains an ORF of 305 amino acids and codes a possibly novel protein. We also estimated the arrangement of sugars by comparing the affinity to various lectin columns.

Climacostol, a defense toxin of the heterotrich ciliate Climacostomum virens against predators

Abstract A toxic substance (climacostol) of the protozoan ciliate Climacostomum virens against the predatory ciliate Dileptus margaritifer was established as 1,3-dihydoxy-5-[(Z)-2′-nonenyl]benzene. The structure was rigorously confirmed by the total synthesis.

Toxic and Phototoxic Properties of the Protozoan Pigments Blepharismin and Oxyblepharismin

Abstract— The toxic and phototoxic properties of blepharismin and oxyblepharismin that were purified from the pigments of a ciliated protozoan, Blepharisma japonicum, by thin‐layer chromatography, were investigated in detail. The toxicity was tested against the ciliated protozoan Dileptus margaritifer, which is relatively sensitive to blepharismin. Although oxyblepharismin has been believed to be neither toxic nor phototoxic, it was found that oxyblepharismin is toxic in the dark and is also phototoxic. This shows that oxyblepharismin can act as a photosensitizer. The toxicity and phototoxicity of these pigments were compared with those of hypericin, which is known to be a typical, strong photosensitizer from plants. It was concluded that blepharismin and oxyblepharismin have strong intrinsic toxicities in the dark compared with hypericin, but their phototoxicities are slightly weaker than that of hypericin. This strong intrinsic toxicity supports our proposal that blepharismin acts as a defensive device against predators in the dark as well as in the light. The decrease in the defensive ability and the increase in the resistance to photokilling of Blepharisma concomitant with its color change from red to blue‐purple in response to weak illumination can be explained by the decrease in toxicity and phototoxicity of the pigment itself and by the decrease in amount of the pigment.

Developmentally and environmentally regulated expression of gamone 1: the trigger molecule for sexual reproduction in Blepharisma japonicum

Sexual reproduction (conjugation) in protozoan ciliates is induced by specific cell-cell interactions between cells of complementary mating types. The ancestral ciliate Blepharisma japonicum has two mating types, I and II. The substances that act as signaling molecules in this extracellular interaction for conjugation are called gamones. The glycoprotein gamone 1, produced by mating type I cells, is a key factor that triggers this interaction. We have previously isolated gamone 1 and determined its complete amino acid sequence. To elucidate the mechanism of initiation of conjugation in ciliates, we investigated the transcription of the gamone 1 gene and found that it is controlled by various internal and external factors. The gamone 1 gene transcript appeared specifically when sexually mature mating type I cells were starved. It was not detected in immature cells, mating type II cells or proliferating cells. The level of transcription was markedly increased in type I cells when they were stimulated with gamone 2, which is secreted by type II cells. This is the first report that the transcription of gamone genes in ciliates is strictly regulated by developmental and environmental factors. This study suggests that the onset of transcription of gamone 1 is linked to the switching mechanism that converts mitotically proliferating cells to differentiated preconjugants, the mechanism of differentiation from immature to mature cells in clonal development, and the mechanism that ensures mating type-specific gene silencing.

Defense function of pigment granules in the ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus (Rhizopodea) and Climacostomum virens (Ciliata).

Abstract The defense function of pigment granules in the red ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus and Climacostomum virens, was investigated by (1) comparing normally-pigmented and albino mutant cells of B. japonicum as the prey of these predators and (2) comparing resistance of the predators to blepharismin, the toxic pigment contained in the pigment granules of B. japonicum. Normally pigmented cells which contained more blepharismin than albino cells were less vulnerable to A. proteus than albino cells, but not to C. virens. C. virens was more resistant than A. proteus to the lethal effect of blepharismin. The results indicate that pigment granules of B. japonicum function as defense organelles against A. proteus but not against C. virens and suggest that successful defense against a predator depends on the susceptibility of the predator to blepharismin.

Behavioural changes induced by the conjugation-inducing pheromones, gamone 1 and 2, in the ciliate Blepharisma japonicum

Preconjugant interactions between complementary mating-type cells in ciliates occur before sexual reproduction. The interactions include retardation of swimming behaviour, courtship dancing, chemoattraction, nuclear activation, cell division, or cell agglutination, depending on ciliate species. In Blepharisma japonicum, chemoattraction of mating-type I by mating-type II has been reported previously. It has been shown that chemoattraction here is caused by a conjugation-inducing substance called gamone 2 secreted by mating-type II cells. In this study, we show that mating-type II cells accumulate near the site where gamone 1 secreted by mating-type I cells is present at a high concentration. We also show that the behaviour of individual cells changes when exposed to the complementary mating-type gamone; cells begin to rotate and swim slowly, thus shortening their minimum path length (final displacement of a cell from its origin). These results suggest that gamones 1 and 2 induce behavioural changes in type II and I cells, respectively, and that gamone-stimulated cells may accumulate at the site with the highest activity of the complementary gamone, after repetition of swimming changes in the gradient of gamone concentration. This reciprocal induction of the changes in behaviour may increase the probability of sexual encounters for conjugation.

Highly divergent actins from karyorelictean, heterotrich, and litostome ciliates

Abstract We have cloned, sequenced, and characterized cDNA of actins from five ciliate species of three different classes of the phylum Ciliophora: Karyorelictea (Loxodes striatus), Heterotrichea (Blepharisma japonicum, Blepharisma musculus), and Litostomatea (Didinium nasutum, Dileptus margaritifer). Loxodes striatus uses UGA as the stop codon and has numerous in-frame UAA and UAG, which are translated into glutamine. The other four species use UAA as the stop codon and have no in-frame UAG nor UGA. The putative amino acid sequences of the newly determined actin genes were found to be highly divergent as expected from previous findings of other ciliate actins. These sequences were also highly divergent from other ciliate actins, indicating that actin genes are highly diverse even within the phylum Ciliophora. Phylogenetic analysis showed high evolutionary rate of ciliate actins. Our results suggest that the evolutionary rate was accelerated because of the differences in molecular interactions.

The Defensive Function of Trichocysts in Paramecium tetraurelia Against Metazoan Predators Compared with the Chemical Defense of Two Species of Toxin-containing Ciliates

The time-honored assumption about the defensive function of trichocysts in Paramecium against predators was recently verified experimentally against different species of unicellular predators. In the present study, we examined the defensive function of trichocysts against three metazoan predators, Cephalodella sp. (Rotifera), Eucypris sp. (Arthropoda), and Stenostomum sphagnetorum (Platyhelminthes). The results confirmed the defensive function of trichocysts against two of these metazoan predators (Cephalodella sp. and Eucypris sp.), while they seem ineffective against S. sphagnetorum. We also compared the defensive efficiency of the trichocysts of P. tetraurelia with that of toxin-containing extrusomes of two ciliates.

Variability of autogamy-maturation pattern in genetically identical populations of Paramecium tetraurelia.

Abstract Autogamy in Paramecium tetraurelia is a form of sexual reproduction in a single cell that results in homozygosity in every genetic locus. Autogamy becomes inducible by natural starvation several fissions after the previous autogamy, and percent autogamy increases gradually with clonal age to reach 100%. We here report the degree of variability of the autogamy-maturation pattern, and how it is inherited through autogamous generations. We assessed the autogamy-maturation pattern by monitoring percent autogamy at the ages of 9, 18 and 27 fissions in the wild-type stock 51. To determine how the autogamy-maturation pattern is inherited, clones that showed the lowest and the highest percent autogamy at age 18 in a given autogamous generation (Gn) were examined for their percent autogamy in the next autogamous generation (Gn+1). This procedure was repeated through successive autogamous generations. We found that percent autogamy at ages 9 and 27 was rather stable (low and high, respectively), while it was extremely variable at age 18 ranging from 3% to 100%. We also found that percent autogamy at age 18 in the progeny clones was variable irrespective of percent autogamy at age 18 in the parental clones; there was no regular rule such as producing progeny with higher (or lower) percent autogamy from parents with lower (or higher) percent autogamy.