Fernando Marvá

Adaptation of the chlorophycean Dictyosphaerium chlorelloides to stressful acidic, mine metal-rich waters as result of pre-selective mutations

Several species of microalgae, closely related to mesophilic lineages, inhabit the extreme environment (pH 2.5, high levels of metals) of the Spains Aguas Agrias Stream water (AASW). Consequently, AASW constitutes an interesting natural laboratory for analysis of adaptation by microalgae to extremely stressful conditions. To distinguish between the pre-selective or post-selective origin of adaptation processes allowing the existence of microalgae in AASW, a Luria-Delbrück fluctuation analysis was performed with the chlorophycean Dictyosphaerium chlorelloides isolated from non-acidic waters. In the analysis, AASW was used as selective factor. Preselective, resistant D. chlorelloides cells appeared with a frequency of 1.1 x 10(-6) per cell per generation. AASW-resistant mutants, with a diminished Malthusian fitness, are maintained in non-extreme waters as the result of a balance between new AASW-resistant cells arising by mutation and AASW-resistant mutants eliminated by natural selection (equilibrium at c. 12 AASW-resistants per 10(7) wild-type cells). We propose that the microalgae inhabiting this stressful environment could be the descendents of chance mutants that arrived in the past or are even arriving at the present.

Adaptation of green microalgae to the herbicides simazine and diquat as result of pre-selective mutations.

Aquatic ecosystems located close to agricultural areas are increasingly polluted by herbicides. We evaluated the capacity for adaptation of green microalgae to lethal concentrations of the herbicide simazine in one strain of Dictyosphaerium chlorelloides and two strains of Scenedesmus intermedius, as well as adaptation to the herbicide diquat in one of the strains of S. intermedius. A Luria-Delbrück fluctuation analysis was carried out in order to distinguish between resistant cells arising from physiological adaptation (acclimatization) or post-adaptive mutation (both events occurring after the exposure to the herbicides), and adaptation due to mutations before the exposure to the herbicides. Simazine-resistant cells arose by rare spontaneous mutations before the exposure to simazine, with a rate of 3.0 x 10(-6) mutants per cell per generation in both strains of S. intermedius, and of 9.2 x 10(-6) mutants per cell per generation in D. chlorelloides. Diquat-resistant cells in S. intermedius arose by pre-selective mutations with a rate of 17.9 x 10(-6) per cell per generation. Rare, pre-selective mutations may allow the survival of green microalgae in simazine- or diquat-polluted waters, via herbicide-resistant selection. Therefore, human-synthesized pollutants, such as the herbicides simazine and diquat, could cause the emergence of evolutionary novelties in aquatic environments.

Toxicity and adaptation of Dictyosphaerium chlorelloides to extreme chromium contamination

Metals are often spilled by industries into inland water environments, with adverse consequences. Numerous papers have reported that heavy metals produce massive destruction of algae. Nevertheless, algal populations seem to become tolerant when they have had previous exposures to heavy metals. Because the mechanisms allowing heavy metal tolerance of algae are not yet known, the present study analyzed the effect of hexavalent chromium on growth and photosynthetic performance of Dictyosphaerium chlorelloides, stressing on the adaptation mechanisms to chromium contamination. Growth and photosynthetic performance of algal cells were inhibited by Cr(VI) at 10 mg/L, and the 72-h median inhibition concentration was established as 1.64 and 1.54 mg/L, respectively. However, after further incubation for a three month period in an environment with 25 mg/L of chromium, some rare, chromium-resistant cells occasionally were found. A Luria-Delbrück fluctuation analysis was performed to distinguish between resistant algae arising from rare, spontaneous mutations and resistant algae arising from physiological adaptation and other adaptive mechanisms. Resistant cells arose only by spontaneous mutations before the addition of chromium, with a rate of 1.77 x 10(-6) mutants per cell division. From a practical point of view, the use of both chromium-sensitive and chromium-resistant genotypes could make possible a specific algal biosensor for chromium.

Inhibition of growth and photosynthesis of selected green microalgae as tools to evaluate toxicity of dodecylethyldimethyl-ammonium bromide

The effect of dodecylethyldimethyl-ammonium bromide (DEAB), a quaternary ammonium, compound widely used as disinfectant, on phytoplankton of inland water systems was analysed by using an experimental model. A toxicity test was based on inhibition of photosynthesis performances (effective quantum yield from photosystem II, ΦPSII and O2 production) of the phytoplanktonic species Scenedesmusintermedius and Dictiosphaerium chlorelloides (Chlorophyceae) under growing doses of DEAB. A concentration-dependent toxic response was obtained in both parameters analysed. In addition, this response was almost immediate. Consequently, the measurement of both parameters (ΦPSII and O2 production) allows to assess DEAB toxicity with higher standards of precision and repeatability. We propose that this procedure could be used to detect presence of quaternary ammonium pollutants in freshwater.

Living in Vulcan's forge: Algal adaptation to stressful geothermal ponds on Vulcano Island (southern Italy) as a result of pre‐selective mutations

Four species of eukaryotic algae proliferate in the sulfureous, acidic (pH 3.1) water of the largest geothermal pond on Vulcano Island (southern Italy). Consequently, this pond constitutes a natural laboratory for analysis of adaptation by phytoplankters to extremely stressful conditions. To distinguish between the pre‐selective or post‐selective origin of adaptation processes allowing the existence of phytoplankters in the pond, a Luria‐Delbrück fluctuation test was carried out with the chlorophycean Dictyosphaerium chlorelloides and the cyanobacterium Microcystis aeruginosa, both isolated from non‐extreme waters; natural water from the Vulcano Island pond was used as selective factor. Preselective, resistant D. chlorelloides cells appeared with a frequency of 4.7 × 10−7 per cell per generation. We propose that the micro‐algae inhabiting this stressful pond could be the descendents of chance mutants that arrived in the past or are even arriving at the present. The genetic adaptation of D. chlorelloides to Vulcano waters could help to explain the survival of photosynthesizers in very stressful geothermal waters during the Neoproterozoic ‘snowball Earth’, a period when primary production collapsed in the biosphere. On the other hand, adaptation to these conditions was not observed in M. aeruginosa, suggesting that cyanobacteria may not be able to develop any kind of adaptation to Vulcano pond water.

Rapid adaptation of some phytoplankton species to osmium as a result of spontaneous mutations

To understand the vulnerability of individual species to anthropogenic contamination, it is important to evaluate the different abilities of phytoplankton to respond to environmental changes induced by pollution. The ability of a species to adapt, rather than its initial tolerance, is the basis for survival under rapidly increasing levels of anthropogenic contamination. High doses of osmium (Os) cause massive destruction of diverse phytoplankton groups. In this study, we found that the coastal chlorophyte Tetraselmis suecica and the continental chlorophyte Dictyosphaerium chlorelloides were able to adapt to a lethal dose of Os. In these species, Os-resistant cells arose as a result of rare spontaneous mutations (at rates of approximately 10−6 mutants per cell division) that occurred before exposure to Os. The mutants remained in the microalgal populations by means of mutation–selection balance. The huge size of phytoplankton populations ensures that there are always enough Os-resistant mutants to guarantee the survival of the population under Os pollution. In contrast, we observed that neither a haptophyte species from open ocean regions nor a cyanobacterium from continental freshwater were able to adapt to the lethal Os dose. Adaptation of phytoplankton to Os contamination is relevant because industrial activities are leading to a rapid increase in Os pollution worldwide.

Microalgal fiber-optic biosensors for water quality monitoring

Novel fiber-optic biosensors have been developed for the analysis of pesticides in water based on Chlorophyceae microalgae immobilized into a porous silicone layer as recognition element, and on measurements of the photogenerated O2 as chemical transducer. The inhibition of O2 production by the photosynthetic green algae in the presence of the pesticide (simazine) was used as the biological signal. Luminescent thin films and a dedicated optoelectronic unit based on emission phase-shift measurements provide the tools for the sensitive O2 measurements. Fluctuation analysis allows selection of sensitive and resistant microalgae mutants without genetic manipulation for maximum biosensing selectivity.