Host and symbiont intraspecific variability: The case of Paramecium calkinsi and "Candidatus Trichorickettsia mobilis".

Newly isolated strains of the ciliate Paramecium calkinsi and their cytoplasmic bacterial endosymbionts were characterized by a multidisciplinary approach, including live observation, ultrastructural investigation, and molecular analysis. Despite morphological resemblance, the characterized P. calkinsi strains showed a significant molecular divergence compared to conspecifics, possibly hinting for a cryptic speciation. The endosymbionts were clearly found to be affiliated to the species "Candidatus Trichorickettsia mobilis" (Rickettsiales, Rickettsiaceae), currently encompassing only bacteria retrieved in an obligate intracellular association with other ciliates. However, a relatively high degree of intraspecific divergence was observed as well, thus it was possible to split "Candidatus Trichorickettsia" into three subspecies, one of which represented so far only by the newly characterized endosymbionts of P. calkinsi. Other features distinguished the members of each different subspecies. In particular, the endosymbionts of P. calkinsi resided in the cytoplasm and possessed numerous peritrichous flagella, although no motility was evidenced, whereas their conspecifics in other hosts were either cytoplasmic and devoid of flagella, or macronuclear, displaying flagellar-driven motility. Moreover, contrarily to previously analyzed "Candidatus Trichorickettsia" hosts, infected P. calkinsi cells frequently became amicronucleate and demonstrated abnormal cell division, eventually leading to decline of the laboratory culture.

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum.

Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria.

Initial steps of infection of the ciliate Paramecium with bacteria Holospora sp.

New light and electron microscope data on the initial steps of endocytobiosis establishment between the ciliate Paramecium and specific intranuclear bacteria Holospora are provided. At the cytoplasmic step of infection bacteria of all Holospora species are found in a vesicle originating from the membrane of the host cell phagosome. The association between host cell microfilaments and the bacterium bearing vesicle may suggest a possible involvement of the ciliate cytoskeleton in the transportation of bacteria to the host cell nucleus. The authors subdivide the process of infection into 6 steps. Some strains of P. caudatum never take up infectious Holospora bacteria in the course of phagocytosis.

Aphidicolin inhibits DNA replication in the micronucleus and blocks cytokinesis in Paramecium caudatum.

Aphidicolin, a selective inhibitor of DNA polymerase, totally blocks DNA replication in the micronucleus but not in the macronucleus of Paramecium caudatum. The ciliates no longer divide and after 4 days the DNA content of the macronucleus has increased by 64%. Concomitantly the cell volume has increased by 53%.

Bacterial endocytobionts of the ciliate Paramecium calkinsi.

Five species of gram-negative bacteria inhabiting the cytoplasm and the macronucleus of the ciliate Paramecium calkinsi are described. Two species of endocytobionts have been found in the cytoplasm. Both of them were represented by only a vegetative form. In the case of the first species, numerous endocytobiotic bacteria (length, 2 µm; diameter, 0.3-0.4 µm) were enclosed in individual endobiontophoral vacuoles. The second one was not as abundant in its host cells and the bacteria lie freely in the host cytoplasm. These bacteria (length, 2-3 µm; diameter, 0.6-0.9 µm) often contained viral particles. Three species of endocytobionts have been discovered in the macronucleus. The first one (1-2 µm long, 0.3-0.4 µm in diameter) resembled Nonospora macronucleata. However, it differed from N. macronucleata in having specific electron dense zones in its cytoplasm and stained differently by Ag-NOR. The second one represents a new species of the Holospora genus: H. curvata sp. nov. The infectious form of this endocytobiont (15-20 µm long, 0.7-0.9 µm in diameter) is curved, the vegetative form (2-3 µm long, the same diameter) was rod-like or slightly curved. The host specificity of these endocytobionts is well expressed. The third species of intranuclear bacteria, H. bacillata, had been previously described in P. woodruffi. The infective capacity of H. curvata and two "stocks" of H. bacillata from different hosts was compared.