Behavioural changes induced by the conjugation-inducing pheromones, gamone 1 and 2, in the ciliate Blepharisma japonicum
Introduction
Conjugation is induced in the ciliate Blepharisma japonicum Suzuki (1954) by interactions between cells of complementary mating-types (I and II) through actions of soluble gamones (Miyake 1968; Miyake and Beyer 1973; Miyake 1981). Gamone 1, secreted by mating-type I cells, is a glycoprotein (Miyake and Beyer 1974). The gamone 1 gene was isolated and its complete amino acid sequence was reported (Sugiura and Harumoto 2001). Gamone 2 is secreted by mating-type II cells and is a non-peptide substance that has been identified as calcium-3- (2′-formylamino-5′-hydroxybenzoil) lactate, a derivative of tryptophan (Kubota et al. 1973) that can be chemically synthesized (Entzeroth and Jaenicke 1981; Entzeroth et al. 1983; Tokoroyama et al. 1973, 1978). When sexually mature mating-type I cells are moderately starved, cells autonomously produce and secrete gamone 1 (Miyake 1981). Gamone 1 specifically acts on mating-type II cells, transforming them so that they can unite, and inducing them to produce and secrete gamone 2. Gamone 2 similarly transforms type I cells so that they can unite and promotes the production of gamone 1. Cells with the capacity to unite can form conjugant pairs.
In some species of ciliates, characteristic cell behaviours are known to occur during the preconjugant cell-cell interactions, such as retardation in swimming, courtship dancing, chemoattraction, nuclear activation, cell division, and cell agglutination (Miyake 1996 for review). In Euplotes raikovi, a typical sexual behaviour is induced by a exogenous pheromone (pheromone released by complementary mating type), and has been studied by electrophysiological methods (Stock et al. 1999).
In B. japonicum, type II cells attract type I cells with gamone 2 (Honda and Miyake 1975; Miyake and Rivola 1989). Gamone 2 can attract type I cells at concentrations as low as 0.001 U/ml (1.3 pM) or lower. The chemoattraction by gamone 2 is considered to base on kinesis (Miyake and Rivola 1989) though there is no experimental evidence. It has been suggested that chemoattraction and induction of cell union are mediated by the same receptor to gamone 2 (Honda and Miyake 1975; Honda 1979), and that the affinity between gamone 2 and its receptor is probably not too high, so that it can sense rapid changes in gamone 2 concentration (Miyake 1996). However, receptors against gamones in B. japonicum have never been identified and still need to be explored thoroughly. Moreover, behavioural response of individual cells under gamone-present conditions has not yet been examined precisely. Whether gamone 1 has an ability to accumulate complementary mating-type cells as well as gamone 2 has been controversial.
In this study, we examined the behavioural response of B. japonicum cells to the gamone released by the complementary mating-type. We clearly show that type II cells also accumulate in the area where gamone 1 concentration is high and that the behaviour of single cells of either mating-type is affected by their exposure to the gamone of the complementary mating-type.
Section snippets
Cells and cell culture
R1072 (mating type I) and R48 (mating type II), which are strains derived from Bangalore strain R, and T121 (mating type II), which originated from the Tsushima strain of B. japonicum, were used. Cells were cultured at 25 °C in WGP (Wheat Grass Powder, Pines) medium (Tokusumi and Takagi 2000), concentrated by mild centrifugation, washed with a physiologically balanced solution (modified SMB, synthetic medium for Blepharisma; designated as SMB below) (Miyake 1981; Sugiura and Harumoto 2001), and
Accumulation of mating-type II cells by mating-type I cells
To examine the ability of gamone 1 to attract mating-type II cells, we placed mating-type I cells in the chamber and mating-type II cells in the beaker outside the chamber (Fig. 1A). We monitored the distribution of cells every 2-3 hours. At the beginning, all cells were distributed evenly inside and outside the chamber. Gamone 1 that was autonomously secreted by type I cells leaked out of the chamber through the window. Outside the chamber, mating-type II cells gradually accumulated at the
Discussion
During the preconjugant interaction in ciliates, cells undergo various behavioural and morphological changes (Miyake 1996). One of these changes is a chemoattraction induced by cells of the complementary mating-type. Ciliates that show chemoattraction in their preconjugant interactions include Euplotes raikovi (Stock et al. 1999), Euplotes octocarinatus (Kuhlmann et al., 1997), Dileptus anser (Afon'kin and Yudin 1987), Tokophrya infusionum (Sonneborn 1978), and probably Euplotes woodruffi (
Acknowledgments
We thank Dr. A. Miyake and Dr. Y. Takagi for helpful advice and discussions. We also thank Prof. H. Iio (Osaka City University, Japan) for providing synthetic gamone 2 and helpful support. This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to T. H. (No. 14580713, No.18570129) and to M. S. (No. 18-714).
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