Modulating effects of orally supplied Euglena gracilis on the physiological responses of the freshwater mussel Diplodon chilensis, exposed to sewage water pollution in a Patagonian river (Argentina).

In order to test if orally supplied Euglena sp. cells modulate the physiological status of bivalves during bioremediation procedures, we evaluated the effect of Euglena gracilis diet on the immune response, oxidative balance and metabolic condition of Diplodon chilensis exposed to sewage water pollution. Mussels were fed for 90 days with E. gracilis (EG) or Scenedesmus vacuolatus (SV, control diet), and then exposed for 10 days at three sites along the Pocahullo river basin: 1) an unpolluted site, upstream of the city (control, C); 2) upstream (UpS) and 3) downstream (DoS) from the main tertiary-treated sewage discharge, in the city of San Martín de los Andes, Northwest Patagonia, Argentina. Our results show that the total hemocyte number decreases while pollution load increases along the river course for both, EG and SV mussels. Phagocytic activity is higher in EG mussels than in SV ones under all conditions. Reactive oxygen species (ROS) production in hemocytes increases with the increase in the pollution load, being significantly higher for EG mussels than for SV ones at DoS; no changes are observed for total oxyradical scavenging capacity (TOSC). Hemocytes' viability is increased for E. gracilis diet at C and remains unchanged in this group of mussels when exposed at the polluted sites. Lysosomal membrane stability is higher in EG mussels than in SV ones for all conditions, although it is decreased at polluted sites compared with that at C. Antioxidant (catalase) and detoxifying (gluthatione S-transferase) defenses are generally lower in gills and digestive gland of EG mussels than in SV ones. Lipid peroxidation (TBARS) is evident in gills of EG mussels at C, and in digestive gland of the same group, at all the sites. Gill mass factor (GF) is affected by the E. gracilis diet; it is increased at C and decreased at polluted sites when compared with that of SV ones. Digestive gland mass factor (DGF) is higher in EG mussels than in SV ones. In D. chilensis, continuous and long term feeding with E. gracilis cells favors immune response and reduces the damage caused by sewage pollution exposure on hemocytes. Nevertheless, diet and transplantation procedures may produce negative effects on the oxidative balance of gills and digestive gland and should be taken into account for bioremediation strategies.

Long-term feeding with Euglena gracilis cells modulates immune responses, oxidative balance and metabolic condition in Diplodon chilensis (Mollusca, Bivalvia, Hyriidae) exposed to living Escherichia coli.

We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells on immune response, oxidative balance and metabolic condition of the freshwater mussel Diplodon chilensis. Mussels, previously fed with Scenedesmus vacuolatus (SV) or E. gracilis (EG) for 90 days, were challenged with an environmentally relevant concentration of Escherichia coli in water for 5 days, under feeding or starvation conditions. EG diet increased overall phagocytic activity and tissue hemocyte accumulation (gill and mantle), and favored hemocyte viability upon E. coli challenge. Tissular hemocyte accumulation, and humoral bacteriolytic activity and protein content were similarly stimulated by EG and E. coli, with no further effect when both stimuli were combined. Both, E. coli challenge and EG diet reduced gill bacteriolytic activity with respect to nonchallenged SV mussels, while no effect was observed in challenged EG mussels. Gill and digestive gland protein contents, along with digestive gland bacteriolytic activity were higher in EG than in SV mussels. Both SV and EG mussels showed increased gill mass upon E. coli challenge, while digestive gland mass was increased by bacterial challenge only in SV mussels. Bacterial challenge produced no effect on humoral reactive oxygen species levels of both groups. Total oxyradical scavenging capacity levels was reduced in challenged SV mussels but remained unaffected in EG ones. In general, EG diet decreased glutathione S-transferase and catalase activities in gill and digestive gland, compared with SV diet; but increased enzyme activity was evident in challenged mussels of both groups. Gill and digestive gland lipid peroxidation levels were higher in EG than in SV mussels but E. coli challenge had stronger effect on SV mussels. Adductor muscle RNA:DNA ratio was higher in EG mussels than in SV ones, and increased upon E. coli challenge in mussels of both groups. E. gracilis can be suggested as a nutritional and protective diet complement suitable for filtering bivalves. However, our results obtained from starved mussels show that starvation periods after supplying this diet should be avoided, since these could revert part of the acquired benefits and/or exacerbate detrimental effects.

Ultrastructural alterations in Phacus brachykentron (Euglenophyta) due to excess of organic matter in the culture medium.

Morphological and ultrastructural changes induced by exposure to excess of organic matter were analyzed in Phacus brachykentron (Pochm.). The cells were isolated from sites in Matanza River, Buenos Aires, Argentina, which have a high degree of organic matter contamination coming from waste waters discharges of the meat industry. Master strains were cultured on soil water medium and a toxicity bioassay was performed. As a result of the enriched medium, several morphological and ultrastructural cellular alterations were observed by optical, scanning, and transmission electron microscopy. Among these, we can point out changes in cell dimensions, remarkable widening of some pellicle bands, increased number and volume of paramylon grains, displacement of the nucleus from the central to the lateral position, some chloroplasts with their thylakoids disordered, and cell lysis. The response to organic enrichment was very fast, i.e. during the 48h of the bioassay. Therefore, any significant increase of organic matter would rapidly affect wild euglenoids. Our results suggest that the alterations observed, such as the presence of large intracellular paramylon bodies or the deformation of euglenoid cells in natural samples, have the potential to be used as environmental bioindicators.

Chromium induced stress conditions in heterotrophic and auxotrophic strains of Euglena gracilis.

Oxidative stress parameter and antioxidant defense compound as well as enzyme activity were studied in relation to different Cr(VI) concentrations (0, 10, 20, 40 µM) in two strains of Euglena gracilis, one isolated from a polluted river (MAT) and the other acquired from a culture collection (UTEX). Chromium toxicity was measured in the auxotrophic and obligated heterotrophic variants of the two strains. Chromium uptake was higher in auxotrophic cultures, reflected by their higher cell proliferation inhibition and lower IC50 levels compared to heterotrophic ones. In the Cr(VI) treatments a reduction of chlorophyll a and b ratio (Chl a/Chl b) was observed, the ratio of protein to paramylon content was augmented, and total lipid content increased, having the auxotrophic strains the highest values. TBARS content increased significantly only at 40 µM Cr(VI) treatment. Unsaturated fatty acids also increased in the Cr(VI) treatments, with the higher storage lipid (saturated acids) content in the heterotrophic cells. The antioxidant response, such as SOD activity and GSH content, increased with chromium concentration, showing the highest GSH values in the heterotrophic cultures and the SOD enzyme participation in chromium toxicity. The MAT strain had higher IC50 values, higher carbohydrate and saturated acid content, and better response of the antioxidant system than the UTEX one. This strain isolated from the polluted place also showed higher GSH content and SOD activity in control cells and in almost all treated cultures. SOD activity reached a 9-fold increase in both MAT strains. These results suggest that tolerance of MAT strain against Cr(VI) stress is not only related to GSH level and/or biosynthesis capacity but is also related to the participation of the SOD antioxidant enzyme.

Trachelomonas (Euglenophyta) from a eutrophic reservoir in Central Mexico.

This study provides valuable information on the ultrastructure and environmental conditions of the Trachelomonas Ehr. (Euglenophyceae) genus in the Guadalupe Dam, a eutrophic reservoir located in the suburbs of Mexico City, which receives a considerable volume of wastewaters. Specimens were collected at surface level between November 2005 and May 2006. Using LM and SEM twelve taxa from phytoplankton were identified of which, 9 are new records for Mexico. The reservoir is warm monomictic, with basic pH values (7.4-10.1), a high concentration of chlorophyll a(18-101 microg l(-1), a permanent anoxic bottom, specific conductivity (K25) of 205 to 290 microS cm(-1), N-NO3, 0.19-1.2 mg l(-1) and P-PO4 0.22-1.6 mg l(-1). Water temperature was 15.6-23.0 degrees C. Most of the Trachelomonas species were found during the dry season, when concentrations of organic matter, nitrogen and phosphorus as well as the temperature were the highest. Higher species richness was also associated with the warmer months. This research contributes to increase our knowledge on Trachelomonas in Mexico and constitutes the first detailed description of lorica ultrastructure of 12 taxa that grow in a body of water with high concentration of nutrients and a moderate amount of mineral contents.

In vivo microspectroscopy monitoring of chromium effects on the photosynthetic and photoreceptive apparatus of Eudorina unicocca and Chlorella kessleri.

In microorganisms and plants, chromium (Cr) is not essential for any metabolic process, and can ultimately prove highly deleterious. Due to its widespread industrial use, chromium has become a serious pollutant in diverse environmental settings. The presence of Cr leads to the selection of specific algal populations able to tolerate high levels of Cr compounds. The varying Cr-resistance mechanisms displayed by microorganisms include biosorption, diminished accumulation, precipitation, reduction of Cr(6+) to Cr(3+), and chromate efflux. In this paper we describe the effects of Cr(6+) (the most toxic species) on the photosynthetic and photoreceptive apparatus of two fresh water microalgae, Eudorina unicocca and Chlorella kessleri. We measured the effect of this heavy metal by means of in vivo absorption microspectroscopy of both the thylakoid compartments and the eyespot. The decomposition of the overall absorption spectra in pigment constituents indicates that Cr(6+) effects are very different in the two algae. In E. unicocca the metal induced a complete pheophinitization of the chlorophylls and a modification of the carotenoids present in the eyespot after only 120 h of exposition at a concentration equal or greater than 40 microM, which is the limit for total Cr discharge established by US EPA regulations. In C. kessleri, chromium concentrations a hundred times higher than this limit had no effect on the photosynthetic machinery. The different tolerance level of the two algae is suggested to be due to the different properties of the mucilaginous envelope and cell wall covering, respectively, the colonies of Eudorina and the single cells of Chlorella, which binds chromium cations to a different extent.

Effects of chromium on photosynthetic and photoreceptive apparatus of the alga Chlamydomonas reinhardtii.

Chromium is a highly toxic non-essential metal for microorganisms and plants. Due to its widespread industrial use, chromium (Cr) has become a serious pollutant in diverse environmental settings. The presence of Cr leads to the selection of algal populations able to tolerate high levels of Cr compounds. The diverse Cr-resistance mechanisms displayed by microorganisms include biosorption, diminished accumulation, precipitation, reduction of Cr(6+) to Cr(3+), and chromate efflux. In this paper we describe the effects of Cr(6+) (the more toxic species) on the photosynthetic and photoreceptive apparatus of the fresh water unicellular alga Chlamydomonas reinhardtii. We measured the effect of the heavy metal by means of in vivo absorption microspectroscopy of both the thylakoid compartments and the eyespot. The decomposition of the overall absorption spectra in pigment constituents indicates that Cr(6+) induced a complete pheophinitization of the chrorophylls and a modification of the carotenoids present in the eyespot only when its concentration is equal or greater than 10 microM. Due to this low tolerance level, C. reinhardtii could be used as indicator of Cr pollution, but it is not feasible for bioremediation purposes.

Gene expression patterns in Euglena gracilis: insights into the cellular response to environmental stress.

To better understand Euglena gracilis gene expression under different stress conditions (Chromium, Streptomycin or darkness), we undertook a survey of the E. gracilis transcriptome by cDNA sequencing and microarray analysis. First, we constructed a non-normalized cDNA library from the E. gracilis UTEX strain and sequenced a total of 1000 cDNAs. Six hundred and ten of these ESTs were similar to either Plantae or Protistae genes (e-value

Effect of chromium on the fatty acid composition of two strains of Euglena gracilis.

The effect of hexavalent chromium on fatty acid composition was studied in two strains of Euglena gracilis; UTEX 753 (from the Culture Collection of Algae of Texas University, USA) and MAT (isolated from a highly polluted River). Both were grown in photoauxotrophic and photoheterotrophic conditions and exposed to two metal concentrations, one below and one above IC50. The high malondialdehyde (MDA) levels (3 to 7-fold) obtained with chromium concentration above IC50, suggested the existence of metal-induced lipid peroxidation. Total lipid content increased only with concentration below IC50, whereas it was inhibited by higher metal concentration. Photoheterotrophic control strains exhibited a significantly higher proportion of saturated and polyunsaturated fatty acids. Polyunsaturated acids were most affected by chromium, especially those related to chloroplast structures. Ultra-structure studies showed clear thylakoid disorganization in all treated cells. The results indicate that hexavalent chromium affects levels of fatty acids, especially those related to photosynthetic activity.

FINE STRUCTURE AND TAXONOMY OF MONOMORPHINA AENIGMATICA COMB. NOV. (EUGLENOPHYTA)(1).

The euglenoid genus Monomorphina was defined by Mereschowsky in 1877 to include rigid euglenoids that were pyriform in lateral view, had a hyaline spine at the posterior end, and one to few parietal chloroplasts typically without pyrenoids. The genus included taxa previously assigned to Phacus Dujardin or Euglena Ehrenberg. The general structure of Monomorphina aenigmatica comb. nov. is described on the basis of light microscopy and scanning and transmission electron microscopy. Cells were pear-shaped in lateral view, rounded at the anterior end and narrowed posteriorly, tapering into a long twisted tail. The pellicle had helically arranged strips spiralled in a counter-clockwise fashion. A distinctive feature of M. aenigmatica was the presence of a single chloroplast bearing a pyrenoid, capped with a paramylon plate. The large parietal chloroplast extended along most of the cell with three prominent cup-shaped paramylon caps on the external face. In transverse section, the chloroplast appeared C-shaped. Because of the ambiguity surrounding the original descriptions used to diagnose this taxon, we designated an epitype for Monomorphina aenigmatica. Morphological features of this species were compared to other members of the genus.

A MOLECULAR ANALYSIS OF THE EUGLENOPHYTES USING SSU RDNA.

Almost since the creation of the genus Euglena (Ehrenberg), the taxa assigned to it have been separated, split apart, and reorganized into new genera based on morphological relationships, resulting in the creation of the genera Phacus (Dujardin), Lepocinclis (Perty), Astasia (Pringsheim), and Khawkinea ( Jahn and McKibben) based on intuitive methods. In an effort to assess the validity of these genera, we have used small subunit (SSU) rDNA data to generate a phylogenetic framework for these genera, with particular attention to the genus Euglena. Using the conserved sequence areas, we performed a phylogenetic analysis using parsimony, maximum likelihood, and distance methods. These different criteria have resulted in trees of the same topology. The euglenoid clade was composed of phagotrophic euglenoids at the base, which gave rise to phototrophs that in turn gave rise to osmotrophs. Among the photosynthetic taxa, the biflagellate form diverged prior to the uniflagellate form. Additionally, the need for a revision in the taxonomy of some of these genera was demonstrated. Currently, taxa from the photosynthetic genera Euglena, Phacus, and Lepocinclis do not form monophyletic clades, but are intermixed with each other as well as with the osmotrophic taxa, Astasia and Khawkinea.