Rapid adaptation of microalgae to bodies of water with extreme pollution from uranium mining: an explanation of how mesophilic organisms can rapidly colonise extremely toxic environments.

Extreme environments may support communities of microalgae living at the limits of their tolerance. It is usually assumed that these extreme environments are inhabited by extremophile species. However, global anthropogenic environmental changes are generating new extreme environments, such as mining-effluent pools of residual waters from uranium mining with high U levels, acidity and radioactivity in Salamanca (Spain). Certain microalgal species have rapidly adapted to these extreme waters (uranium mining in this area began in 1960). Experiments have demonstrated that physiological acclimatisation would be unable to achieve adaptation. In contrast, rapid genetic adaptation was observed in waters ostensibly lethal to microalgae by means of rare spontaneous mutations that occurred prior to the exposure to effluent waters from uranium mining. However, adaptation to the most extreme conditions was only possible after recombination through sexual mating because adaptation requires more than one mutation. Microalgae living in extreme environments could be the descendants of pre-selective mutants that confer significant adaptive value to extreme contamination. These "lucky mutants" could allow for the evolutionary rescue of populations faced with rapid environmental change.

Estimating the capability of different phytoplankton groups to adapt to contamination: herbicides will affect phytoplankton species differently.

• Investigating the differential capacity of the response of phytoplankton to human-induced environmental forcing has become a key issue to understanding further the future repercussions on the functioning of aquatic ecosystems. • The initial tolerance to the widely dispersed herbicide simazine was measured in diverse phytoplankton species. An experimental ratchet system maintaining large populations of dividing cells (which ensures the occurrence of rare spontaneous mutations that confer adaptation) and a strong selection pressure (which ensures the preservation of such mutations within the population) was later applied to estimate the capability of different groups of phytoplankton to adapt to simazine. • Initially, simazine doses between 0.05 and 0.15 ppm were able to inhibit 100% growth in all the species tested. However, a significant increase in simazine resistance was achieved in all derived populations during the ratchet experiment. The differential capacity for simazine adaptation was observed among the different species. • The capacity of different species to adapt to simazine can be explained in relation to taxonomic group, ploidy, growth rate and habitat preference. Haploid populations of continental Chlorophyta showed the greatest capacity to adapt to simazine. By contrast, populations of Haptophyta of open ocean regions were the group least capable of adapting to the herbicide.

Toxic effects and specific chromium acquired resistance in selected strains of Dyctiosphaerium chlorelloides.

Due to its various uses, chromium contamination has become widespread in a diverse array of environments. The present study was carried out to investigate the toxic effect of chromium exposures on sensitive and resistant strains of the green algae Dyctiosphaerium chlorelloides, and to determine the nature and mechanism of chromium-resistant cells that arise. The toxic effect on the photosynthetic performance of chromium exposures in both cell populations, and the sensitive differences due to chromium oxidation state, were estimated, and the results indicate that although the photosynthetic performance in both strains were inhibited, there are not significant differences among IC(50(72)) values obtained in toxicity assays with both chromium oxidation states in wild-type cells, and however these differences are very significant when the assays were performed with Cr(VI) resistant cells. The 72-h 50% inhibitory concentration values obtained with Cr(III) exposures were similar for both strains. Additionally, by means of the SEM/EDX and TEM microscopic techniques, the occurrence of rapid morphological evolution in the microalgal cells and the possible detoxificant mechanisms was observed after exposure of the wild strain to chromium hexavalent. Moreover, the different response in photosynthetic activity observed between sensitive and resistant cells of D. chlorelloides in the presence of Cr(VI) and Cr(III) could be used to obtain a chromium-specific eukaryotic microalgal biosensor.

Microalgae response to petroleum spill: an experimental model analysing physiological and genetic response of Dunaliella tertiolecta (Chlorophyceae) to oil samples from the tanker Prestige.

In November 2002, the oil tanker Prestige sank off the northwestern coast of Spain, spilling more than 50,000 tons of petroleum with disastrous ecological and economical consequences. In order to analyse the harmful consequences of the oil spill on marine microalgae, short- and long-term effects of oil samples from the Prestige spill were studied using laboratory cultures of Dunaliella tertiolecta (strain Dt1Lwt). Significant inhibition of photosynthesis (assessed by F(v)/F(m), ETR(max) and alpha estimations) was observed after only 1h of oil exposure with clear concentration dependency. Three days later, photosynthetic activity remained inhibited although cell survival was only slightly effected. In cultures exposed to the lowest oil concentration, mitotic rates and percentage of motile cells were 17-33% and 12-42% of the controls, respectively. After 1 month, neither dividing nor motile cells were observed at the highest oil concentrations. However, after further incubation, occasionally the growth of rare cells resistant to oil was found. A fluctuation analysis was carried out to distinguish between resistant cells arising from rare spontaneous mutations and resistant cells arising from physiological or other mechanisms of adaptation. The existence of rapid evolution as result of preselective mutations from petroleum sensitivity to petroleum resistance was observed. Resistant cells arose by rare spontaneous mutations prior to the addition of oil, with a mutation rate of 2.76x10(-5) oil-resistant mutants per cell division. Apparently, rare spontaneous preselective mutations are able to assure the survival of microalgae in oil-polluted environments.

Toxic effect and adaptation in Scenedesmus intermedius to anthropogenic chloramphenicol contamination: genetic versus physiological mechanisms to rapid acquisition of xenobiotic resistance.

Anthropogenic water pollution is producing a challenge to the survival of phytoplankton populations. From an ecological point of view, the tolerance of these microorganisms to water pollution is of paramount importance since they are the principal primary producers of aquatic ecosystems. The adaptation of a common chlorophyta species (Scenedesmus intermedius) exposed to selected dose-response chloramphenicol (CAP) concentrations has been analyzed. A fluctuation analysis demonstrated that CAP-resistant cells arise due to spontaneous mutation which occurs randomly prior to the antibiotic exposure. CAP-inhibited growth and photosynthetic performance of algal cells at 0.28 mg/l, and the IC(50(72)) value was established in 0.10 mg/l for both parameters. The mutation rate from CAP sensitivity to resistance was 1.01 x 10(-5) mutations per cell division, while the frequency of CAP-resistant allele in non-polluted environment was estimated to be 5.5 CAP-resistant mutants per 10(3) sensitive-cells. These results demonstrate that resistant mutants exhibit a diminished fitness until 5 mg/l of CAP, thus enabling the survival of microalgae population.

Microcystins from tap water could be a risk factor for liver and colorectal cancer: a risk intensified by global change.

An increasing number of people drink water from fresh water supply reservoirs. However, with the global change a lot of reservoirs become eutrophic, which facilitates the occurrence of toxin-producing cyanobacterial blooms. Microcystins (powerful hepatotoxic water-soluble heptapeptides) are the most important cyanobacterial toxins affecting humans. High doses of microcystins produce hepatic necrosis. Consequently, WHO Guidelines limit microcystins to 1 ppb in drinking waters. However, microcystins are present frequently in tap water at lower doses. Here, we hypothesized that chronic consume of tap water containing low doses of microcystins may be a risk factor for liver and colorectal cancer. Two kinds of evidences support this hypothesis. On one hand some epidemiological data (mainly in China). On the other hand, the molecular mechanism of microcystins toxicity (inhibition of protein phosphatases PP1 and PP2) is just like okadaic acid (a potent tumor promoter). Cancer risk from drinking water is certainly less than smoking, occupational exposures or some foods. But it is significant and with a rapid increase of toxic cyanobacterial blooms by eutrophycation, become more frequent.

Copper sulphate and DCMU-herbicide treatments increase asymmetry between sister cells in the toxic cyanobacteria Microcystis aeruginosa: implications for detecting environmental stress.

Works correlating fluctuating asymmetry with environmental stress or genetic damages have been largely reported in multicellular organisms but not in single-celled ones. We hypothesize that asymmetry analysis could also be applied to single-celled organisms, because the asymmetry between two sister cells originated from a cellular division event (same genotype in similar environment) must tend to zero in the absence of environmental or genetic perturbations. Laboratory experiments with copper sulphate and DCMU-herbicide treatments as well as experiments in a water reservoir after treatment with copper sulphate algaecide show that environmental stress increases asymmetry between sister cells of Microcystis aeruginosa (Cyanobacteria). Even low Cu(2+) or DCMU doses, which were unable to reduce growth rate, considerably enlarge asymmetry with respect to untreated controls. Asymmetry between sister cells of cyanobacteria seems to be a reliable indicator of environmental perturbation. Analysis of asymmetry in single-celled organisms could become as important as fluctuating asymmetry of multicellular organisms is today.

Detection of infectious Cryptosporidium parvum oocysts in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule).

Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 10(3) oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.

Preference of mice to consume Microcystis aeruginosa (toxin-producing cyanobacteria): a possible explanation for numerous fatalities of livestock and wildlife.

Cyanobacteria often produce severe illness and in some cases spectacular fatality on livestock and wildlife world-wide. Heavy cyanobacterial waterblooms usually form patches of dense surface scum, and terrestrial animals drinking such concentrated dirty froth can consume a fatal dose. Surprisingly, animals do not avoid swallowing concentrated microbial scum. Different experiments of drink selection were performed with laboratory mice to determine why animals drink these concentrated scum. These experiments showed that animals elected to consume dense cultures of the toxic cyanobacteria Microcystis aeruginosa in preference to limpid water. When M. aeruginosa cells were supplied ad libitum, mice avidly swallowed these toxic cyanobacteria until this led to their death. Mice were unable to detect the phycotoxin (microcystin). In contrast, mice did not select cultures containing other non-toxic phytoplanktonic organisms. Observations in nature suggest that this preference in the consumption of toxic cyanobacteria is common among other animal species.

Revised dinoflagellate phylogeny inferred from molecular analysis of large-subunit ribosomal RNA gene sequences.

The nucleotide sequence analysis of the PCR products corresponding to the variable large-subunit rRNA domains D1, D2, D9, and D10 from ten representative dinoflagellate species is reported. Species were selected among the main laboratory-grown dinoflagellate groups: Prorocentrales, Gymnodiniales, and Peridiniales which comprise a variety of morphological and ecological characteristics. The sequence alignments comprising up to 1,000 nucleotides from all ten species were employed to analyze the phylogenetic relationships among these dinoflagellates. Maximum parsimony and neighbor-joining trees were inferred from the data generated and subsequently tested by bootstrapping. Both the D1/D2 and the D9/D10 regions led to coherent trees in which the main class of dinoflagellates. Dinophyceae, is divided in three groups: prorocentroid, gymnodinioid, and peridinioid. An interesting outcome from the molecular phylogeny obtained was the uncertain emergence of Prorocentrum lima. The molecular results reported agreed with morphological classifications within Peridiniales but not with those of Prorocentrales and Gymnodiniales. Additionally, the sequence comparison analysis provided strong evidence to suggest that Alexandrium minutum and Alexandrium lusitanicum were synonymous species given the identical sequence they shared. Moreover, clone Gg1V, which was determined Gymnodinium catenatum based on morphological criteria, would correspond to a new species of the genus Gymnodinium as its sequence clearly differed from that obtained in G. catenatum. The sequence of the amplified fragments was demonstrated to be a valuable tool for phylogenetic and taxonomical analysis among these highly diversified species.

The use of lectins to characterize and separate living canine spermatozoa: A preliminary study.

This study was designed as a preliminary attempt to develop a methodology for relating the glucidic structure of the sperm membrane to sperm morphology. Differences in plasma membrane glycoconjugates between motile and nonmotile spermatozoa were studied by using 7 lectins. Fresh spermatozoa from 3 dogs were analyzed by fluorescein isothiocyanate (FITC) labeled lectins. The binding of lectins to the sperm membrane and the capability of the lectins to agglutinate spermatozoa were estimated semi-quantitatively by observation with either an epifluorescence or a phase contrast microscope, respectively. All the lectins tested bound to non motile spermatozoa, with Helix pomatia , Pisum sativum and Arachis hypogaea showing intense fluorescence, Triticum vulgare and Glycine maxima showing moderate fluorescence, and Phaseolus vulgaris and Phytolacca americana showing low fluorescence. However, Helix pomatia . and Triticum vulgare also bound to rapid and slow moving spermatozoa, and were the only 2 lectins that induced sperm agglutination. These results suggest that lectins could be a possible tool for characterizing and separating spermatozoa with different rates of motility.

Contact Inhibition: Also a Control for Cell Proliferation in Unicellular Algae?

According to traditional views, the proliferation of unicellular algae is controlled primarily by environmental conditions. But as in mammalian cells, other biological mechanisms, such as growth factors, cellular aging, and contact inhibition, might also control algal proliferation. Here we ask whether contact inhibition regulates growth in several species of unicellular algae as it does in mammalian cells. Laboratory cultures of the dinoflagellate Prorocentrum lima (Ehrenberg) Dodge show contact inhibition at low cell density, so this would be an autocontrol mechanism of cell proliferation that could also act in natural populations of P. lima. But, Synechocystis spp., Phaeodactylum tricornutum (Bohlin), Skeletonema costatum (Greville), and Tetraselmis spp. do not exhibit contact inhibition in laboratory cultures because they are able to grow at high cellular density. Apparently their growth is limited by nutrient depletion or catabolite accumulation instead of contact inhibition. Spirogyra insignis (Hassall) Kutz, Prorocentrum triestinum Schiller, and Alexandrium tamarense (Halim) Balech show a complex response, as they are able to grow in both low and high cell density medium. These results suggest that contact inhibition is more adaptative in benthic unicellular algae.

Persistence of cell division synchrony in Spirogyra insignis (Gamophyceae): membrane proteoglycans transmitting synchronizing information throughout generations.

Spirogyra insignis shows a long-term persistence of cell division synchrony in the absence of the synchronizing Zeitgeber, so that at least six generations are involved in the process. This tentatively suggests that a mechanism of transmission throughout generations of synchronizing information could maintain this synchrony. Apparently, a vital part of the molecular basis of this mechanism is a membrane proteoglycan complex. This complex could obtain temporal information from a synchronizing Zeitgeber and be transmitted to the progeny by distribution of plasma membrane between daughter cells.