Heat shock protein of the Hsp70 family in the euryhaline cilate Paramecium nephridiatum and its role in adaptation to salinity changes

The level of the Hsp70 heat shock protein was studied in the cells of euryhaline ciliates Paramecium nephridiatum after environmental salinity changes. Two types of treatment were used: “shock” and “adaptation.” In the former case the ciliates were placed for 1 h into medium with stress salinity, and subsequently returned for 2 h to the medium they were acclimated to. In the latter case the ciliates were placed for 3 h into the medium with the stress level of salinity. The ciliates acclimated to fresh water (0‰ salinity) were shown to have a higher constitutive level of Hsp70 than those acclimated to 10‰. Transfer of the protists from fresh water to medium with 10‰ salinity (the shock medium) did not increase Hsp70 synthesis, whereas the return transfer resulted in induction of Hsp70 in the cells. In both directions of salinity change, “adaptation” led to induction of Hsp70. The obtained results support the hypothesis that salinity of 10‰ is less harmful for the eurihaline ciliate P. nephridiatum, than fresh water is. We also presume that the ability of euryhaline ciliates to survive in a wide salinity spectrum might be determined by the higher constitutive level of their Hsp70 in comparison with that of stenohaline representatives of the same genus.     

Alterations of the level of 70 kDa family heat shock protein in the ciliate Tetrahymena pyriformis in the process of the cells adaptation to the medium salinity changes

Alterations of Hsp70 level were studied in the cells of freshwater ciliate Tetrahymena pyriformis after medium salinity changes. It is shown that ciliates, acclimated to fresh water (0 per thousand) and to salt water of 2 and 10 per thousand have similar constitutive levels of Hsp70 in their cells. Neither pronounced induction of Hsp70, was not decrease of its level, revealed in ciliates after salinity stresses. These data differ from the results obtained while studying euryhaline ciliate Paramecium nephridiatum and strongly freshwater one Paramecium jenningsi. We presume that the differences in the mode of chaperone system reaction of these ciliates species might be connected with different extents of salinity persistence - the least in P. jenningsi, intermediate in T. pyriformis and the most pronounced in P. nephridiatum.     

Morphological and molecular investigations of Paramecium schewiakoffi sp. nov. (Ciliophora, Oligohymenophorea) and current status of distribution and taxonomy of Paramecium spp.

Paramecium schewiakoffi sp. nov. is described from a pond in Shanghai, China. It is a freshwater species belonging to the “aurelia” subgroup of the genus. It is of similar size and shape to P. jenningsi, but has a single large micronucleus of the “chromosomal” morphological type, while P. jenningsi has two smaller micronuclei. The general morphology, morphometric characteristics and nuclear reorganization pattern, a random amplified polymorphic DNA (RAPD) fingerprint pattern, and the small subunit rRNA gene sequence are presented for the species. Comparison of P. schewiakoffi with the other species of Paramecium indicates that it is a valid new species of the genus. Geographical locations reported for many Paramecium species do not support the theory that all ciliates have a cosmopolitan distribution. It is proposed that, in an extension of Jankowski's earlier suggestion, the genus Paramecium should be subdivided into four subgenera: Chloroparamecium, Helianter, Cypriostomum and Paramecium, on the basis of morphometric, biological and molecular differences.     

Heat shock protein of Hsp70 in Paramecium nephridiatum and its role in adaptation to environmental salinity changes

The level of Hsp70 was studied in the cells of eurihaline ciliate Paramecium nephridiatum after the environmental salinity changes. Two types of treatment were applied. "Shock": ciliates were placed for 1 h to the medium with stress salinity, then transferred back to the medium, they were acclimated to, for 2 h; "adaptation": ciliates were placed for 3 h into stress salinity. It has been shown, that ciliates, acclimated to fresh water (0%) have the higher constitutive level of Hsp70, than those, acclimated to 10%. Transfer from fresh water to 10% does not cause the increase of Hsp70 synthesis in protists, whereas the reciprocal transfer results in induction of Hsp70 in the cells. "Adaptation" results in induction of Hsp70 in both "directions" of salinity changes. The results obtained allow to presume that the possibility to survive in the media of various salinity in eurihaline ciliates is somehow determined by the higher initial level of Hsp70 in their cells, than in stenohaline representatives of the same genus.     

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.     

Correlations between salinity-persistence of ciliate species and their constitutive heat shock protein of 70 kDa contents

Our own studies of alterations in the level of constitutive heat shock protein of 70 kDa family (Hsp70) in freshwater (Paramecium jenningsi), meta-freshwater (Tetrahymena pyriformis) and curyhaline (P. nephridiatum) ciliates acclimated to salt-water and fresh-water medium were reviewed. It has been shown that the level of constitutive Hsp70 content correlates with the salinity-resistance of ciliate species: in P. jenning i it was lower in freshwater, than in marine water, in euryhaline P. nephridiatum it was higher in freshwater, than in marine water, and was more or less stable in T. pyriformis, the ciliate that occupies intermediate position between two mentioned above species according its salinity-resistance. The ecological importance of constitutive heat shock protein level is discussed in the context of salinity adaptations studies.     

Rotifers as predators on small ciliates

Clearance rates of Synchaeta pectinata, Brachionus calyciflorus and Asplanchna girodi on Tetrahymena pyriformis (46 µm in length) at a density of 10 cells ml^–1, in the presence of algal food, were 2.5 to 6.1 ml rot.^–1 day^–1. Clearance rates of these rotifers were, respectively, about 2, 3, and 13 times lower on Strobilidium gyrans (58 µm in length) than on T. pyriformis, indicating that the saltations of S. gyrans are an effective escape response. Clearance rates of S. pectinata were considerably lower on Colpidium striatum (81 µm) than on S. gyrans, suggesting that S. pectinata may not be able to ingest ciliates of this size. S. pectinata had a clearance rate of 19 ml rot.^–1 day^–1 on S. gyrans at a density of 1.2 cells ml^–1, in the absence of edible algal food. Rotifers may prey extensively on ciliates in natural plankton communities, ingesting 25 to 50 individuals in the 45–60 µm size range day^–1.     

Stress responses and metal tolerance of <Emphasis Type="Italic">Chlamydomonas acidophila</Emphasis> in metal-enriched lake water and artificial medium

Chlamydomonas acidophila faces high heavy-metal concentrations in acidic mining lakes, where it is a dominant phytoplankton species. To investigate the importance of metals to C. acidophila in these lakes, we examined the response of growth, photosynthesis, cell structure, heat-shock protein (Hsp) accumulation, and metal adsorption after incubation in metal-rich lake water and artificial growth medium enriched with metals (Fe, Zn). Incubation in both metal-rich lake water and medium caused large decreases in photosystem II function (though no differences among lakes), but no decrease in growth rate (except for medium + Fe). Concentrations of small Hsps were higher in algae incubated in metal-rich lake-water than in metal-enriched medium, whereas Hsp60 and Hsp70A were either less or equally expressed. Cellular Zn and Fe contents were lower, and metals adsorbed to the cell surface were higher, in lake-water-incubated algae than in medium-grown cells. The results indicate that high Zn or Fe levels are likely not the main or only contributor to the low primary production in mining lakes, and multiple adaptations of C. acidophila (e.g., high Hsp levels, decreased metal accumulation) increase its tolerance to metals and permit survival under such adverse environmental conditions. Supposedly, the main stress factor present in the lake water is an interaction between low P and high Fe concentrations.     

Identification of Chlorella viruses in Paramecium bursaria clones by pulse-field electrophoresis

The ciliates Paramecium bursaria contain endosymbiotic green algae Chlorella spp. in their cytoplasm. The algae isolated from P. bursaria are sensitive to large DNA-containing viruses of the family Phycodnaviridae. The type virus of this family is PBCV-1 (Paramecium bursaria Chlorella virus). Investigation of the total DNA of P. bursaria clones by pulse-field electrophoresis (PEGE) revealed a pronounced band on PEGE profiles of some P. bursaria clones; the band was formed by DNA molecules of approx. 300 kb. This band probably contained the DNA of Chlorella virus. Two approaches were used in the present work to confirm this hypothesis. Microbiological tests were used to scan a collection of P. bursaria clones for specific types of viruses; the 300-kb band was revealed only in the PEGE profiles of virus-containing clones. Blot hybridization of P. bursaria total DNA separated by pulse-field electrophoresis with the virus-specific probe revealed that the band under study was formed by the DNA of a Chlorella virus. Paramecium clones were shown to contain approx. 10⁵ copies of nonintegrated viral DNA.     

Global molecular variation of Paramecium jenningsi complex (Ciliophora,Protista): a starting point for further,detailed biogeography surveys

The world’s second stand of P. primjenningsi (Paramecium jenningsi complex, Ciliophora, Protista), heretofore known only from India, has been revealed in Ethiopia (Africa). This finding has enlarged the range of this cryptic species and was a trigger to re-analyse the distribution of all members of the complex (known from ～20 tropical locations). The current survey is an initial one, where, based on haplotype networks, a detailed analysis of the relationship within the P. jenningsi complex has been performed. Although the V4 hypervariable fragment of the SSU rDNA gene is widely used as a first-step barcode marker for microbial HTS analyses, it has provided inconclusive results based on the dataset investigated. However, the ITS1-5.8S-ITS2-5’rDNA and COI mtDNA fragments indicate the possibility of the delimitation of the cryptic species of P. jenningsi (which is crucial from the point of view of metabarcoding surveys). We suppose that future sampling of unexplored, tropical regions will certainly change our knowledge about Paramecium biodiversity and biogeography. This sampling will probably rely on the integration of metabarcodes from environmental DNA studies, with molecular data obtained from identified representatives of particular cryptic species.     

Paramecium calkinsi andP. putrinum (Ciliophora, Protista) harboring alpha-subgroup bacteria in the cytoplasm

Summary New intracellular bacteria were detected in the cytoplasm ofParamecium calkinsi andP. putrinum. Some of the bacteria were not evenly distributed in the cytoplasm of the host but were found in the center of the cell, eventually near the nuclei, but not in the cortex area, whereas another species was found in the cortex area. These peculiarities of intracellular bacteria localization in the host suggest that the conditions in various parts of the cytoplasm favor bacterial maintenance to different extent. Due to the results obtained by transmission electron microscopy and in situ hybridization using appropriate oligonucleotide probes, the bacteria, three or possibly four species, are Gram-negative and belong to the alpha-subgroup of proteobacteria. Bacteria from one stock ofP. calkinsi were found to be infectious for bacteria-free cells ofP. calkinsi andP. nephridiatum.     

The “<Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis>” complex of cryptic species

Cryptic species are common among protists and have long been known in ciliates. The ciliate genus Tetrahymena contains a large group of morphologically indistinguishable species referred to as the ‘T. pyriformis’ complex. These species include those reproductively isolated by mating type as well as asexual species characterized by the absence of the germinal micronucleus. This paper examines the molecular diversity of the species and describes the biogeography of ‘T. pyriformis’ species. Most species are globally distributed, though the best studied species, T. thermophila, is confined to North America and gives evidence of population structure in local populations. Selfers and asexual species are common and arise from sexual species, a possible exploitation of nuclear dimorphism. It is argued that the cryptic species likely have different ecological roles and that the biodiversity of Tetrahymena in particular, and ciliates in general, is underestimated. Special Issue: Protist diversity and geographic distribution. Guest editor: W. Foissner.     

Intestinal ciliate composition found in the feces of the Turk rahvan horse Equus caballus, Linnaeus 1758

Species composition and distribution of large intestinal ciliates were investigated in the feces from 15 Turk rahvan horses, living in the vicinity of Izmir, Turkey. Twenty-two ciliate genera consisting of 36 species were identified. This is the first report on intestinal ciliates in Turk rahvan horses and no previously unknown species were observed. The mean number of ciliates was 14.2 ± 13.9 × 10⁴ cells ml⁻¹ of feces and the mean number of ciliate species per host was 9.9 ± 7.1. No ciliates were observed in 2 horses. Bundleia and Blepharocorys were considered to be the major genera since these ciliates were constantly found in high proportions. In contrast, Paraisotricha, Didesmis and Gassovskiella were only observed at low frequencies. The ciliates found in this survey had almost the same characteristics as those described in previous reports, suggesting that there was no significant geographic variation in the intestinal ciliate fauna of equids.     

The effect of environmental salinity on the level of heat shock proteins in gill epithelium of Mytilus edilis L. mussel

The composition and the level of heat shock proteins in the gill epithelium cells of mussels Mytilus edulis L. from the White Sea under different levels of environmental salinity were studied by the method of immunoblotting. In mussels maintained under normal salinity (26%), constitutive Hsp70 and protein of about 40 kDa were revealed. After long-term (11?C14 days) acclimation to 14 and 35?? of the level Hsp70 in gill epithelium cells increased. Hsp70 induction was also observed in cells of isolated gills after salinity shock at 14% for 3 and 24 h.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: Modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015–15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg⁹ to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.     

Production of methane and hydrogen by anaerobic ciliates containing symbiotic methanogens

Rates of methane production by three anaerobic ciliates containing symbiotic methanogens (the marine Metopus contortus and Plagiopyla frontata, and the limnic Metopus palaeformis) were quantified. Hydrogen production by normal (containing active symbionts), aposymbiotic and BES-treated cells was also measured in the case of the marine species. Methanogenesis was closely coupled to host metabolism and growth; at maximum ciliate growth rates (20°C) each methanogen produced about 1 fmol CH₄ per hour corresponding to about 7, 4 and 0.35 pmol per ciliate per hour for M. contortus, P. frontata and M. palaeformis, respectively. Normal cells produced traces of H₂. Hydrogen production by BES-treated or aposymbiotic cells accounted for 75 and 45% of the methane production of normal M. contortus and P. frontata cells, respectively. However, it is possible that hydrogen production was partly inhibited in the absence of methanogens. Theoretical considerations suggest that hydrogen transfer is significant to the metabolism of larger anaerobic ciliates. Ciliates with methanogens produced CH₄ under microaerobic conditions due to their ability to maintain an anoxic intracellular environment at low external oxygen tensions. Methanogenesis was still detectable at a pO₂ of 0.63 kPa (3 %atm sat).     

Further studies of the encapsulation of eggs ofMetaphycus spp. (Hym.: Encyrtidae) by the pyriform scale,Protopulvinaria pyriformis (Hom.: Coccidae)

The encapsulation of eggs ofMetaphycus swirskii Annecke &; Mynhardt (Hymenoptera: Encyrtidae) by the pyriform scale,Protopulvinaria pyriformis (Cockerell) (Homoptera: Coccidae), collected in the coastal plain of Israel, was determined during April 1986 to May 1987. The rates of encapsulation were low in November (13.0%) and relatively high in April, May, August and September (32.0–89.0%). The seasonal variations in the encapsulation of eggs ofM. stanleyi Compere and/orM. swirskii byP. pyriformis infesting avocado (Persea americana) and jambolan (Syzygium cumini) were studied at Miqwe Yisra'el (coastal plain) during October 1986 to February 1988. Encapsulation rates were similar in scales infesting either of the two host plants. They were highest during July to August (49.0–75.0%) and lowest during December to February (0.9–10.0%). Encapsulation incidence at Miqwe Yisra'el was correlated with the ambient temperatures (r=0.89). The rate of encapsulation of parasitoid eggs (M. stanleyi and/orM. galbus Annecke) recorded inP. pyriformis sent to Israel from Spain in September 1987 was 42.2%. The high rates of encapsulation ofMetaphycus spp. eggs byP. pyriformis during the summer, may interfere with efficient biological control of the pest.     

Three-locus analysis in conjunction with strain crosses confirms the existence of reproductively isolated populations in Paramecium jenningsi

Paramecium jenningsi (Diller & Earl, 1958) was formerly considered to be a species with only one syngen (genetic species) based on an inter-strain cross of two strains, cytological analysis, and an investigation of esterases and acid phosphatases. However, the existence of syngens within the species was later suggested by genetic studies, i.e. classical strain crosses of new strains and molecular PCR-based analyses (RAPD, RFLP), as well as by sequencing the H4 gene fragment. This issue still needs to be clarified by the application of molecular markers, genetic tests and cytological preparations. In the present study, we tested 12 strains of P. jenningsi originating from Asia, North America and Africa. Trees reconstructed on the basis of three genome fragments (ITS1-5.8S-ITS2-5’LSU, COI and CytB) show that P. jenningsi is divided into two distinct clusters (PJ1, PJ3) and one branch (PJ2) which correspond to reproductively isolated groups revealed by strain crosses. A study based both on strain crosses and a three-locus comparison gives the opportunity for a more complete identification of the reproductively isolated populations of P. jenningsi and other ciliate species.