Hans-Werner Kuhlmann

Interspecific Morphogens Regulating Prey-Predator Relationships in Protozoa

The ciliate Euplotes octocarinatus and some close relatives of it are triggered by predator-released substances to undergo morphogenetic changes that inhibit their engulfment. The changes occur within a few hours and do not require cell division. They are perpetuated during reproduction so long as the concentration of the morphogen is maintained. The ability of Euplotes to respond to predator-produced signals by a defensive change in cell architecture probably provides an effective mechanism for damping population oscillations ofboth prey and predators andfosters coexistence. The signal-induced cell transformation merits study for its own sake because of its developmental implications.

Predation risk of typical ovoid and ‘winged’ morphs of Euplotes (Protozoa, Ciliophora)

Freshwater species of the genus Euplotes (Protozoa, Ciliophora) change their morphology in the presence of some of their predators. The ciliates develop extended lateral ‘wings’ as well as dorsal and ventral projections which make engulfment by predators more difficult. In a series of laboratory experiments ingestion rates of four protozoan predators, the ciliates Lembadion bullinum, Dileptus anser, Stylonychia mytilus and Urostyla grandis, and one metazoan predator, the turbellarian Stenostomum sphagnetorum, on three species of Euplotes (E. octocarinatus, E. patella and E. aediculatus) were determined. It was calculated that the probability of rejection by a predator changed from 1:1 for ovoid morphs of Euplotes to about 2:1–20:1 for ‘winged’ morphs of Euplotes, dependent on the prey and predator species that were combined. The nutritional condition of the prey also had some influence. In mixed-species cultures of prey and predators, transformed cells of E. octocarinatus survived for several months.

The pheromones and pheromone genes of new stocks of the Euplotes octocarinatus species complex

Summary Two new stocks of the hypotrichous ciliate Euplotes octocarinatus isolated in Florida and Maryland/USA were identified as members of mating type VI (mt 2 mt 3 ), secreting pheromones 2 and 3 and mating type VII (mt 1 mt 1 ), secreting pheromone 1. These are two out of the ten already known mating types of Euplotes octocarinatus. Results of breeding experiments suggest that the new stocks belong to new syngens of this species complex. The analysis of the macronuclear molecules containing the new pheromone 1, 2 and 3-encoding genes show that their structure is very similar to those isolated before. Like in these genes the coding regions are interrupted by three introns with a high degree of homology. A comparison of the deduced amino acid sequences of the new pheromones with the known versions of the corresponding pheromones reveals homologies of 76 to 93% for the secreted proteins whereas the leader sequences are 98 to 100% identical. In both versions of pheromones 1, 2 and 3 there are ten cysteine residues which are located at strictly conserved positions. In spite of their differences both versions of pheromones 1, 2 and 3 show the same specific biological activity. This might be an indication that the unchanged residues participate in receptor binding.

PHEROMONES OF THE CILIATE EUPLOTES OCTOCARINATUS NOT ONLY INDUCE CONJUGATION BUT ALSO FUNCTION AS CHEMOATTRACTANTS

Cells of the ten mating types of the ciliate Euplotes octocarinatus communicate by pheromones before they enter conjugation. The pheromones induce homotypic pairing when applied to mating types that do not secrete the same pheromone(s). Heterotypic pairs (i.e., those between cells of different mating types) are formed only when both mating types in a mixture secrete a pheromone that the other does not. The genetics of mating types is based on four codominant mating type alleles, each allele determining production of a different pheromone. Here we report that the pheromones not only induce pair formation but also attract cells. This was shown by placing cells of various mating types in neighboring agar wells so that the pheromones could diffuse from one well to the next. We found that the cells accumulated on the side of the well where a pheromone entered by diffusion. This response was observed only if the pheromone had the capacity to induce the cells to conjugate. That the pheromones and not some other substances attract the cells was shown by placing pheromone 3, expressed in Escherichia coli, in wells next to tester strains. Mating types known to respond to pheromone 3 by pair formation also showed accumulation on the side of the well at which the pheromone entered by diffusion. Since the pattern of cell attraction corresponds with the pattern of conjugation induction, we suggest that not only conjugation induction but also cell attraction is governed by pheromone-specific receptors. In addition, we describe a succession of changes in the behavior of cells affected by the pheromones.

Nuclear processes in Euplotes octocarinatus during conjugation

The pregamic and metagamic nuclear divisions during conjugation of Euplotes octocarinatus and the development of new micro- and macronuclei were investigated with the help of phase contrast microscopy and a staining of the cells by aceto carmine. The most important stages are illustrated with microphotographs and a time table of the events based on experiments carried out at 26°C is presented. It is shown that E. octocarinatus has n = 35 chromosomes and that all of them enter the macronuclear anlage and undergo polytenisation before they break down into the short DNA-pieces typical of the adult macronucleus of hypotrich ciliates. The nuclear events suggest that, similar to what has been reported for E. patella, frequently sister nuclei become the pronuclei which should result in the formation of a high proportion of isogenic co-conjugant lines. A comparison of the nuclear events in heterotypic pairs (co-conjugants of different mating types) with those in homotypic pairs (co-conjugants of the same mating type) revealed no differences apart from a tendency of homotypic pairs to become arrested at the premeiotic division and then to separate. Although hundreds of cells were investigated, regeneration of pieces of the old macronucleus or fusion of fragments with a newly formed anlage, as is reported for certain other Euplotes species, has not been observed.

Escape response of Euplotes octocarinatus to turbellarian predators

Summary Physical contact between the freshwater ciliate Euplotes octocarinatus and its turbellarian predator, Stenostomum sphagnetorum , elicits a behavioural response of the potential prey which is different from the normal “avoiding reaction” of Euplotes . Because of its defensive character it is called “escape response”. The response was analysed by the use of a video camera and recorder system. It was found that the escape response takes a regular course, beginning with sudden backward moving of Euplotes , followed by a rapid turn around and a subsequent forward movement. Velocities during the escape response are 1 to 5 times higher than calculated for control cells. The whole reaction lasts about 2 s. Within this time a Euplotes cell moves more than 1,000 µm away from its predator. While 90% of well-fed Euplotes cells demonstrated the defensive response, starved cells cultivated separately from their predators revealed fewer escape responses, however, several of the starved cells regained the capacity for the escape response after exposure to Stenostomum or Stenostomum -conditioned medium for 20 h. It is discussed whether or not the inducible behavioural response is triggered by the same predator-released substances that are known to induce defensive morphological changes in Euplotes .

Giants in Lembadion bullinum (Ciliophora, Hymenostomata) - General Morphology and Inducing Conditions

Summary The freshwater hymenostome Lembadion bullinum exists in two forms: “normal” cells and “giants”. The general morphology of both morphotypes was studied, biometrical data for both morphs are presented and compared with each other. Beside a sharp increase in cell length and width, giants are characterized by their larger oral region, by an increased number of meridional kineties, by a larger macronucleus and in certain stocks by the occurrence of additional micronuclei. It is shown that the majority of giants present in a culture originates in binary fission of already transformed cells. The ex novo transformation of normal cells into giants regularly occurred in starved cultures as a consequence of cannibalism. At higher cell densities (≥ 250 cells/ml) relatively more individuals transformed into giants than at low cell densities (≤ 50 cells/ml). There is no evidence for any soluble or cell bound “giant inducing factor” in this species.

Do phototactic ciliates make use of directional antennas to track the direction of light

Summary Phototaxis describes the directed movement of motile microorganisms with respect to the direction of light. In ciliates, phototactic orientation has been reported for more than a dozen species. Some, like Stentor and Blepharisma , arc brightly coloured due to their numerous pigment granules. In Chlamydodon , a stigma appears in conjunction with a behavioural change from negative to positive phototaxis. The histophagous ciliates Ophryoglena and Porpostoma , which either possess a watchglass organelle or a composed crystalline organelle, display different phototactic behaviour at different stages of their life cycles. It has been suggested that the characteristic pigments and cell organelles of phototactic ciliates play important roles in the detection of the light direction. Bleached cells with a reduced number of pigment granules, transparent cells without a stigma, and cells that lacked a watchglass organelle due to micromanipulation were found to be little sensitive to light, or showed inaccurate or no phototactic orientation. While the pigment granules of the heterotrichs presumedly contain the photoreceptor molecules mediating photo-orientation, the stigma of Chlamydodon , the watchglass organelle of Ophryoglena , and the composed crystalline organelle of Porpostoma are suspected shading or light reflecting structures. Interacting with the sites of sensory transduction, which should be located in the cell membrane nearby, but are unidentified, conspicuous organelles could be used by the cells as direction-sensitive light detectors.

Phototaxis in Porpostoma notatum, a marine scuticociliate with a composed crystalline organelle

Summary The histophagous scuticociliate Porpostoma notatum passes through five developmental stages during its life cycle: theronts, trophonts, protomonts, tomonts and tomites. The stages are characterized by both, a different morphology and behaviour. When cells are exposed to radiant white light of 10 klx, negative phototaxis occurs in trophonts, which represent the feeding stage of the reproduction cycle, as well as in protomonts and young tomonts, the two successive stages after food uptake. Tomites, resulting from one or two divisions of a tomont, show no phototactic orientation until they have differentiated into theronts. Theronts may show either a positive or negative phototactic response. Individuals of all life cycle stages, except for young tomites, commonly bear a well-developed cup-like organelle, which faces the oral cavity with its concave side. This organelle is composed of alternating layers of cytoplasm and crystals. By this architecture, the organelle resembles a multilayer interference reflector, a structure which has been described in multicellular organisms several times. A possible function of the crystalline organelle in photo-orientation of P. notatum is discussed.

Interspecific mating reactions between Euplotes octocarinatus and Euplotes patella syngen 2

Euplotes octocarinatus and E. patella syngen 2 were tested for their capacity to undergo interspecific mating reactions. Pheromones which the cells of both species secrete to induce cells of other mating types of the same species to prepare for conjugation by changing their cell surface properties were found to act only species-specific. Cells which have changed their surface properties under the influence of their species-specific pheromones, however, are able to form interspecific conjugant pairs. Our cytological investigations show that the micronuclear processes of about 75% of interspecific conjugant pairs become arrested in the course of the premeiotic division. In these cases the co-conjugants separate from each other and return to the vegetative cell cycle. About 25 % of the interspecific pairs enter meiosis, but the nuclear processes in the co-conjugants are not well synchronized and finally become blocked at metaphase of the first meiotic division. The fate of such co-conjugants is always cell death. The results show that sexual incompatibility between the two species is not only due to different pheromones; in addition, it appears to be necessary that the progress of the nuclear events in a cell is stimulated repeatedly by species-specific signals provided by the co-conjugant.