Effects of antibiotics on the early infection process of a macronuclear endosymbiotic bacterium Holospora obtusa of Paramecium caudatum

We examined the effects of antibiotics involved in bacterial DNA, RNA and protein synthesis and host protein synthesis on the early infection process of the bacterium Holospora obtusa, a macronucleus-specific symbiont of the ciliate Paramecium caudatum. Infection of the host macronucleus by the bacterium was not inhibited by mitomycin C, rifampicin and chloramphenicol. However, ingestion of the bacterium into the host digestive vacuoles and escape of the bacterium from the vacuoles to the host cytoplasm were significantly arrested with emetine. The results suggest that newly synthesized host proteins play an important role in the early infection process.     

Scanning Electron Microscopic Observation of Differentiation from the Reproductive Short Form to the Infectious Long Form of Holospora obtusa

To identify the surface features of Holospora obtusa during its differentiation from the reproductive short form to the infectious long form, bacteria of four different buoyant densities were isolated by Percoll density gradient centrifugation of homogenates of host cells or isolated macronuclei, and examined with a scanning electron microscope. Bacteria of buoyant density 1.09 g/ml were reproductive short forms as well as cells at various stages in the elongation process including fully elongated ones. Bacteria of buoyant densities 1.11 g/ml and 1.13 g/ml were premature long forms and those of 1.16 g/ml were mature infectious long forms. Bacteria of buoyant density 1.09 g/ml had an entirely rough surface while those of buoyant densities 1.11 g/ml and 1.13 g/ml were smooth and had wale-like stripes on their surface. A small tapered tip was observed at one end of the bacteria of buoyant density 1.13 g/ml. Bacteria of buoyant density 1.16 g/ml had an entirely smooth surface, but one end always showed a rough surface; this locally differentiated surface of the special tip of the infectious long form may be responsible for both the nuclear and species specificities of the infectivity of H. obtusa. These observations indicate that the surface of H. obtusa changes during differentiation and the special tip develops in bacteria of buoyant density 1.13 g/ml.     

Method for the Simultaneous Establishment of Many Axenic Cultures of Paramecium*†

SYNOPSIS. A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmasterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing ～ 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliates can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock each of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

Experimental evolution of local parasite maladaptation

Sign and magnitude of local adaptation in host–parasite systems may vary with ecological, epidemiological or genetic parameters. To investigate the role of host genetic background, we established long‐term experimental populations of different genotypes of the protozoan Paramecium caudatum, infected with the bacterial parasite Holospora undulata. We observed the evolution of an overall pattern of parasite local maladaptation for infectivity, indicating a general coevolutionary disadvantage of this parasite. Maladaptation extended to host populations with the same genetic background, similar to extending from the local to a higher regional level in natural populations. Patterns for virulence were qualitatively similar, but with less statistical support. A nonsignificant correlation with levels of (mal)adaptation for infectivity suggests independent evolution of these traits. Our results indicate similar (co)evolutionary trajectories in populations with different genetic backgrounds. Nonetheless, the correlated clines of genetic distance and parasite performance illustrate how genetic background can shape spatial gradients of local adaptation.     

Evolution of specialization in a spatially continuous environment

We study the evolution of specialization in a spatially continuous (one‐dimensional) environment divided into two habitats; we use a general trade‐off function relating fitnesses in the two habitats and illustrate our results with two classical trade‐off functions. We show that the population can either reach an intermediate value of the trait and be moderately adapted to both habitats (1 generalist), or split into two locally adapted subpopulations (2 specialists). We recover the qualitative results obtained with simpler metapopulation models with island migration: the evolutionary outcome depends on the concavity of the trade‐off, on the proportion of each habitat and on migration. Our quantitative prediction on migration, however, depends on isolation by distance. Our spatially explicit model may thus be particularly useful to describe the evolutionary dynamics of specialization in, perhaps, more realistic ecological scenarios.     

Macronuclear Regeneration and Cell Division in Paramecium caudatum

SYNOPSIS. In Paramecium caudatum , occurrence of macronuclear regeneration is closely related to the time of feeding after conjugation. Macronuclear regeneration is induced with a high frequency when conjugating pairs are transferred into fresh culture medium. Feeding immediately after conjugation induces early cell division and 3 or more fissions occur without macronuclear division because of the inability of the macronuclear anlagen to divide. In the cells lacking normal macronuclear anlagen, old macronuclear fragments undergo regeneration and form vegetative macronuclei.     

Ciliary Membranes and Mating Substances in Paramecium caudatum

SYNOPSIS Cilia detached from mating reactive cells of Paramecium caudatum were fractionated for the purpose of identifying the structural component bearing mating substances. Purified axoneme fractions had no mating reactivity. The membrane fraction obtained by dialyzing against a solution of Tris-EDTA (0.1 m m EDTA, 1 m m Tris-HCI, pH 7.6) and 0.6 m KCI, and then by centrifuging over 40% (w/v) sucrose was strongly reactive. No mating reactivity was detected in the soluble fractions containing axonemal and matrix proteins. The results indicate that the mating substances in active from are localized only on the ciliary membranes.     

Fate of the 63-kDa periplasmic protein of the infectious form of the endonuclear symbiotic bacterium Holospora obtusa during the infection process

Holospora obtusa is a macronucleus-specific endosymbiotic bacterium of the ciliate Paramecium caudatum. We report the secretion of a 63-kDa periplasmic protein of an infectious form of the bacterium into the macronucleus of its host. Indirect immunofluorescence microscopy with five monoclonal antibodies against the 63-kDa protein demonstrated that, soon after the bacterial invasion into the host macronucleus, the protein was detected in the infected macronucleus and that levels of the protein increased dramatically within one day of infection. The use of inhibitors for host and bacterial protein synthesis illustrated that, in early infection of H. obtusa, not only the pre-existing but also a newly synthesized 63-kDa protein was secreted into the host macronucleus. A partial amino acid sequence of the protein was determined, and a gene encoding the 63-kDa protein was cloned. The deduced amino acid sequence shows that this protein is a novel protein.     

Temperature-Sensitive Behavior of Paramecium caudatum

SYNOPSIS. The effect of temperature on the behavior of swimming cells of Paramecium caudatum has been investigated by photographic analyses of their tracks in uniform temperature, in temperature gradient, or in temperature changing with time. When the cells were placed in the temperature gradient, the frequency of discontinuous directional changes of cells swimming toward the optimal temperature, the temperature of the culture, was much lower than that of the cells swimming in the opposite direction. This difference in the frequency of directional changes explained the observed accumulation of the cells at - the optimal temperature. When the temperature was suddenly changed toward the optimum, a transient decrease of the frequency of directional changes was observed and when the temperature was changed in the reverse direction, a transient increase of the frequency was noted. This transient response to the temperature change was the origin of the dependence of the frequency of directional changes on the swimming direction in the temperature gradient. Finally, the relation between the magnitude of the transient response and the rate of the temperature change was derived.     

Intraspecific genetic variation in Paramecium revealed by mitochondrial cytochrome C oxidase I sequences

Studies of intraspecific genetic diversity of ciliates, such as population genetics and biogeography, are particularly hampered by the lack of suitable DNA markers. For example, sequences of the non-coding ribosomal internal transcribed spacer (ITS) regions are often too conserved for intraspecific analyses. We have therefore identified primers for the mitochondrial cytochrome c oxidase I (COI) gene and applied them for intraspecific investigations in Paramecium caudatum and Paramecium multimicronucleatum. Furthermore, we obtained sequences of the ITS regions from the same strains and carried out comparative sequence analyses of both data sets. The mitochondrial sequences revealed substantially higher variation in both Paramecium species, with intraspecific divergences up to 7% in P. caudatum and 9.5% in P. multimicronucleatum. Moreover, an initial survey of the population structure discovered different mitochondrial haplotypes of P. caudatum in one pond, thereby demonstrating the potential of this genetic marker for population genetic analyses. Our primers successfully amplified the COI gene of other Paramecium. This is the first report of intraspecific variation in free-living protozoans based on mitochondrial sequence data. Our results show that the high variation in mitochondrial DNA makes it a suitable marker for intraspecific and population genetic studies.     

Relation Between Cell Union and Nuclear Reorganization in Triplet Conjugants of Paramecium caudatum and Paramecium multimicronucleatum

SYNOPSIS Triplet conjugants of Paramecium caudatum which appeared naturally in mating mixtures and those of Paramecium multimicronucleatum which were produced by conjugation-inducing chemicals were isolated. Triplet conjugants lasting for more than 3 h were stained to examine macronuclear events. In P. caudatum , only 2 triplets among 182 (1%) contained macronuclear fragmentation in all 3 members. The most frequently occurring triplets (79%) were those producing 1 cell without and 2 cells with macronuclear fragments. There were also triplets (17%) producing 1 cell with, and 2 without macronuclear fragments, and some (3%) with 3 cells that contained no fragments. The length of persistence of the triplet was not responsible for the occurrence of macronuclear fragmentation in the 3rd cell of the triplet. In P. multimicronucleatum , the same 4 classes of triplets occurred, but the most frequently occurring class was that consisting of 3 cells (91%) with macronuclear fragments. Induction of nearly 100% of triplets with 3 such cells was possible by isolating the triplets' from a culture which was treated chemically at about 24 h after the last feeding. Treatment with chemicals in starved cultures resulted in triplets with incompletely fragmented or nonfragmented macronuclei. Further, in P. multimicronucleatum , chemicallyinduced triplets involving only holdfast pairs to which the 3rd cells were uniting often produced 3 cells with fragmented macronuclei.