UV Radiation and Visible Light Induce hsp70 Gene Expression in the Antarctic Psychrophilic Ciliate Euplotes focardii.

The psychrophilic ciliate Euplotes focardii inhabits the shallow marine coastal sediments of Antarctica, where, over millions of years of evolution, it has reached a strict molecular adaptation to such a constant-temperature environment (about -2 °C). This long evolution at sub-zero temperatures has made E. focardii unable to respond to heat stress with the activation of its heat shock protein (hsp) 70 genes. These genes can, however, be expressed in response to other stresses, like the oxidative one, thus indicating that the molecular adaptation has exclusively altered the heat stress signaling pathways, while it has preserved hsp70 gene activation in response to other environmental stressors. Since radiative stress has proved to be affine to oxidative stress in several organisms, we investigated the capability of UV radiation to induce hsp70 transcription. E. focardii cell cultures were exposed to several different irradiation regimes, ranging from visible only to a mixture of visible, UV-A and UV-B. The irradiation values of each spectral band have been set to be comparable with those recorded in a typical Antarctic spring. Using Northern blot analysis, we measured the expression level of hsp70 immediately after irradiation (0-h-labeled samples), 1 h, and 2 h from the end of the irradiation. Surprisingly, our results showed that besides UV radiation, the visible light was also able to induce hsp70 expression in E. focardii. Moreover, spectrophotometric measurements have revealed no detectable endogenous pigments in E. focardii, making it difficult to propose a possible explanation for the visible light induction of its hsp70 genes. Further research is needed to conclusively clarify this point.

Understanding UV-driven metabolism in the hypersaline ciliate Fabrea salina.

By using NMR spectroscopy, a non-invasive investigation technique, we performed in vivo experiments aimed at uncovering the metabolic pathways involved in the early response of Fabrea salina cells to ultraviolet (UV) radiation. This hypersaline ciliate was chosen as a model organism because of its well-known high resistance to UV radiation. Identical cell samples were exposed to visible radiation only (control samples, CS) and to UV-B + UV-A + visible radiation (treated samples, TS), and NMR spectra of in vivo cells were collected at different exposure times. Resonances were identified through one- and two-dimensional experiments. To compare experiments performed at variable irradiation times on different culture batches, metabolite signals affected by the UV exposure were normalized to corresponding intensity at τ = 0, the zero exposure time. The most affected metabolites are all osmoprotectants, namely, choline, glycine-betaine, betaines, ectoine, proline, α-trehalose and sucrose. The time course of these signals presents qualitative differences between CS and TS, and most of these osmoprotectants tend to accumulate significantly in TS in a UV dose-dependent manner. A picture of the immediate stress response of F. salina against UV radiation in terms of osmoprotection, water retention and salting-out prevention is described.

In vivo NMR metabolic profiling of Fabrea salina reveals sequential defense mechanisms against ultraviolet radiation.

Fabrea salina is a hypersaline ciliate that is known to be among the strongest ultraviolet (UV)-resistant microorganisms; however, the molecular mechanisms of this resistance are almost unknown. By means of in vivo NMR spectroscopy, we determined the metabolic profile of living F. salina cells exposed to visible light and to polychromatic UV-B + UV-A + Vis radiation for several different exposure times. We used unsupervised pattern-recognition analysis to compare these profiles and discovered some metabolites whose concentration changed specifically upon UV exposure and in a dose-dependent manner. This variation was interpreted in terms of a two-phase cell reaction involving at least two different pathways: an early response consisting of degradation processes, followed by a late response activating osmoprotection mechanisms. The first step alters the concentration of formate, acetate, and saturated fatty-acid metabolites, whereas the osmoprotection modifies the activity of betaine moieties and other functionally related metabolites. In the latter pathway, alanine, proline, and sugars suggest a possible incipient protein synthesis as defense and/or degeneration mechanisms. We conclude that NMR spectroscopy on in vivo cells is an optimal approach for investigating the effect of UV-induced stress on the whole metabolome of F. salina because it minimizes the invasiveness of the measurement.

Optimizing soluble protein extraction and two-dimensional polyacrylamide gel electrophoresis quality for extremophile ciliates.

An efficient protein extraction methodology is quite important for sample preparation and subsequent 2-D PAGE and MS analysis. Cell lysis is the first step in protein extraction and purification. Many techniques are available for cell disruption, including physical and detergent-based methods. Here, we report on a very fast and efficient detergent-free Tris-based method to extract the soluble fraction proteins of extremophile ciliates, comparing it with a detergent-based protocol. This comparison has been carried out by means of 2-D PAGE and subsequent MALDI-compatible silver staining of protein samples obtained from the intensely pigmented hypersaline ciliate Fabrea salina and the Antarctic hypotrich ciliate Euplotes focardii. Our results indicate that this fast and easy extraction method allows to obtain more clear crude extracts and more spot-abundant polyacrylamide gels.

Evidence for ciliary pigment localization in colored ciliates and implications for their photosensory transduction chain: a confocal microscopy study.

In this study we report for the first time the localization of a photoreceptor pigment in the cilia of the colored heterotrich ciliates Blepharisma japonicum red and blue form, Fabrea salina, and Stentor coeruleus, as result of a confocal microscopy investigation. Optical sectioning confocal microscopy has been used for studying the spatial distribution of the pigment in the cell body, surprisingly showing that, besides its expected presence in the cortical region immediately below the cell membrane, it is located in the cilia too. In order to ascertain possible differences in the pigment fluorescence properties along the cell body, we have measured emission spectra from different parts of it (anterior, posterior, and cilia). Our results clearly indicate that in all cases the spectra are the same, within experimental errors. Finally, we have evaluated the pigment relative fluorescence efficiency of these ciliates. In an ordered scale from lower to greater efficiency, we have S. coeruleus, B. japonicum blue, B. japonicum red, and F. salina. The possible implications of our findings for the process of photosensory transduction are discussed.

Biological weighting function of the UV-B-induced impairment of phototaxis in the freshwater ciliate Ophryoglena flava.

We determined the biological weighting function (BWF) of the effect of UV radiation on phototaxis of the freshwater, histophagous ciliate Ophryoglena flava. Dose-effect curves were measured by exposing the cells to 12 different irradiation regimens obtained with two different levels of UV-B radiation and by using six filters with cutoff wavelengths ranging from 280 to 335 nm. The results show that there are significant damages to phototaxis at the doses used and that the effect increases when the cutoff is shifted toward short wavelengths. The data were used to calculate the BWF of phototaxis impairment by applying a nonlinear fit procedure. The BWF thus obtained decays exponentially with increasing wavelength in agreement with similar findings reported in the literature for other systems.

Effects of UV-B irradiation on a marine microecosystem.

Purpose of this work was to study the effect of UV irradiation on a microecosystem consisting of several interacting species. The system chosen was of a hypersaline type, where all the species present live at high salt concentration; it comprises different bacteria; a producer, the photosynthetic green alga Dunaliella salina; and a consumer, the ciliated protozoan Fabrea salina, which form a complete food chain. We were able to establish the initial conditions that give rise to a self-sustaining microecosystem, stable for at least 3 weeks. We then determined the effect of UV irradiation on this microecosystem under laboratory-controlled conditions, in particular by measuring the critical UV exposure for the two main components of the microecosystem (algae and protozoa) under UV-B irradiances comparable to those of solar irradiation. In our experiments, we varied irradiance, total dose and spectral composition of the actinic light. The critical doses at irradiances of the order of 56 kJ/m(2) (typical average daily irradiance in a sunny summer day in Pisa), measured for each main component of the microecosystem (algae and ciliates), turned out to be around 70 kJ/m(2) for ciliates and 50 kJ/m(2) for D. salina. By exposing microecosystems to daily UV-B irradiances of the order of 8 kJ/m(2) (typical average daily irradiance in a sunny winter day in Pisa), we found no effect at total doses of the order of the critical doses at high irradiances, showing that the reciprocity law does not hold. We have also measured a preliminary spectral-sensitive curve of the UV effects, which shows an exponential decay with wavelength.

Effect of preillumination on photomotile responses of the marine ciliate Fabrea salina.

Fabrea salina is a marine ciliate that shows photomotile responses such as positive phototaxis and a step-down photophobic reaction. We found that preilluminated F. salina cells show a phototactic response significantly greater than that of dark-adapted cells when exposed to the same phototactic light stimulus. In particular, positive phototaxis is strongly enhanced by preillumination. This enhancement effect depends on the preillumination light irradiance, on the total preillumination dose, and on the duration of the dark interval between preillumination and the phototaxis measurement. Our results show that the determining factor is the total preillumination dose given to the sample. The enhancement effect shows an asymptotic behavior over a certain range of energy values (10-200 W/m2). Further, the effect is transient; after 120 s in the dark, the cells lose any memory of the preillumination, independent of the preillumination energy received. These results are tentatively discussed in terms of light-driven membrane potential or membrane channel conductances.