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La presencia de biotoxinas en los moluscos es largamente conocida y ampliamente vigilada. En España, las dos que más se han detectado en los últimos años han sido las saxitoxinas y el ácido okadaico (toxinas PSP y DSP, respectivamente). A efecto de evitar intoxicaciones agudas en la población, existen unos límites máximos de toxinas que pueden estar presentes en el alimento de venta al consumidor. Sin embargo, la presencia de toxina a concentraciones inferiores a la legalmente establecida puede producir intoxicaciones crónicas o efectos a largo plazo. El objetivo del estudio es detectar la presencia de toxinas que están llegando a consumo humano, estén o no dentro del límite de concentración permitido. Se realizó un muestreo en diferentes pescaderías de la Comunidad de Madrid, sin incluir la propia ciudad de Madrid, y se analizó la concentración de toxinas PSP y DSP presentes en 50 muestras de mejillones, almejas, berberechos, vieiras y zamburiñas. Los resultados indican que un 4% de las muestras de los moluscos adquiridos contenían saxitoxinas y en un 6% se detectó ácido okadaico, ya sea en forma de trazas o con una positividad confirmada en base al método analítico, si bien los datos obtenidos cumplen los límites máximos establecidos a nivel comunitario.

El cambio ambiental global incrementa la proliferación de cianobacterias y la liberación de cianotoxinas muy peligrosas en aguas de abastecimiento y recreo. Estas citotoxinas pueden ocasionar graves problemas de salud, por lo que se hace necesario incrementar el control, especialmente en las aguas de baño, para evitar eventos indeseables en la población. Se analizaron cianobacterias productoras de toxinas y cianotoxinas (microcistinas) en muestras de columnas agua y de la superficie de las zonas de baño autorizadas por la Comunidad de Madrid, así como en embalses para abastecimiento de agua de consumo. Los muestreos se realizaron en los meses desde mayo a octubre de 2018. De cada lugar se realizaron, al menos, dos determinaciones en diferentes tiempos. Con excepción de la Playa de Presillas (Rascafría), en las zonas muestreadas se han detectado cianobacterias productoras de cianotoxinas, así como niveles de microcistina mayores de 1μg/L, considerado perjudicial para la salud. Los máximos tanto de microcistinas como de cianobacterias se encuentran en los meses de julio y agosto. El estudio confirma que hay presencia de microcistinas en concentraciones tóxicas en época de baño. Se hace necesario establecer controles de las cianotoxinas estableciendo un sistema de aviso de aparición de los blooms tóxicos en aguas de recreo.

Lithium isotopes are essential for nuclear energy, but new enrichment methods are required. In this study, we considered biotechnology as a possibility. We assessed the Li fractionation capabilities of three Chlorophyte strains: Chlamydomonas reinhardtii, Tetraselmis mediterranea, and a freshwater Chlorophyte, Desmodesmus sp. These species were cultured in Li containing media and were analysed just after inoculation and after 3, 12, and 27 days. Li mass was determined using a Inductively Coupled Plasma Mass Spectrometer, and the isotope compositions were measured on a Thermo Element XR Inductively Coupled Plasma Mass Spectrometer. The maximum Li capture was observed at day 27 with C. reinhardtii (31.66 µg/g). Desmodesmus sp. reached the greatest Li fractionation, (δ6 = 85.4‰). All strains fractionated preferentially towards 6Li. More studies are required to find fitter species and to establish the optimal conditions for Li capture and fractionation. Nevertheless, this is the first step for a microalgal nuclear biotechnology.

In the year 2020 COVID-19 pandemic was a global issue that changed mankinds lifestyle. Since then, when we will control the pandemic and recover our normal life has become the paramount question to be answered, and it needs to be solved. One problem is that there are wealthy countries, with very good health care systems and scientific resources while others barely dedicate 100 US $ per citizen per year, rich countries could cooperate at different levels with poorer ones. In such a diverse context classic epidemiology models, excellent for predicting short term evolution of the pandemic at a local level are not as suitable for long term predictions at a global scale specially if the data they use are of questionable accuracy. Alternatively, big data and AI approaches have been tried. There is an option that can be more effective. Physics applies predictive models about the duration of an event based on analysing the dynamics of the time evolution of the event itself. These models can be used alongside with probabilistic and game theory models that consider different degrees of cooperation. By means of the physics Delta-targument and a game theory model (cooperate versus defector) we calculate when different countries may control COVID-19 pandemic. In a non-cooperate model, those countries with more resources and best manage the pandemic will have it under control between May and September 2021, whereas those with no resources will suffer the pandemic until at least October 2023. On the other hand, a strong cooperative model will allow that the majority could control the COVID-19 pandemic between October 2021 and November 2022.

Rare Earth Elements (REE) increasing demand prompts the research of biotechnological approaches to exploit secondary resources. We made use of the adapted Fluctuation analyses experiment to obtain Chlamydomonas reinhardtii ChlA strains resistant to Samarium (Sm) as the reference REE. The starting hypothesis was that adaptation to metal-containing media leads to an enhanced metal uptake. ChlA was able to adapt to 1.33·10−4 Sm M and pH~3 by pre-existing genetic variability, allowing the evolutionary rescue of 13 of the 99 populations studied. The rescuing resistant genotypes presented a mutation rate of 8.65·10−7 resistant cells per division. The resulting resistant population contradicted the expected fitness cost associated with the adaptation to Sm, selection resulted in larger and faster-growing resistant cells. Among the three isolated strains studied for Sm uptake, only one presented uplifted performance compared to the control population (46.64 μg Sm gˉ¹ of wet biomass and 3.26·10−7 ng Sm per cell, mainly bioaccumulated within the cells). The selection of microalgae strains with improved tolerance to REEs by this methodology could be a promising solution for REES sequestration. However, increased tolerance can be independent or have negative effects on uptake performance and cellular features studied are not directly correlated with the metal uptake. Summary sentence Repurposing a classic laboratory evolution experiment to select for microalgae Samarium adapted strains for metals recovery and biotechnology approaches. Data availability statement All data generated or analyzed during this study are included in this published article (and its raw files).

Using data from 50 very different countries (which represent nearly 70% of world’s population) and by means of a regression analysis, we studied the predictive power of different variables (mobility, air pollution, health & research, economic and social & geographic indicators) over the number of infected and dead by SARS-CoV-2. We also studied if the predictive power of these variables changed during a 4 months period (March, April, May and June). We approached data in two different ways, cumulative data and non-cumulative data.The number of deaths by Covid-19 can always be predicted with great accuracy from the number of infected, regardless of the characteristics of the country.Inbound tourism emerged as the variable that best predicts the number of infected (and, consequently, the number of deaths) happening in the different countries. Electricity consumption and air pollution of a country (CO2emissions, nitrous oxide and methane) are also capable of predicting, with great precision, the number of infections and deaths from Covid-19. Characteristics such as the area and population of a country can also predict, although to a lesser extent, the number of infected and dead. All predictive variables remained significant through time.In contrast, a series of variables, which in principle would seem to have a greater influence on the evolution of Covid-19 (hospital bed density, Physicians per 1000 people, Researches in R & D, urban population…), turned out to have very little - or none- predictive power.Our results explain why countries that opted for travel restrictions and social withdrawal policies at a very early stage of the pandemic outbreak, obtained better results. Preventive policies proved to be the key, rather than having large hospital and medical resources.

Resulting from the nuclear fuel cycle, large amounts of depleted uranium (DU) tails are piling up, waiting for possible use or final disposal. To date, the recovery of the residual 235U isotope contained in DU has been conducted only marginally by physical processes. Relative isotope abundances are often mediated by biological processes, and the biologically driven U isotopic fractionation has been previously identified in reducing bacteria. Our results indicate that the cells of two microalgal strains (freshwater Chlamydomonas sp. (ChlGS) and marine Tetraselmis mediterranea (TmmRU)) took up DU from the exposure solutions, inducing U isotopic fractionation with a preference for the fissile 235U isotope over 238U. The n(235U)/n(238U) isotopic fractionation magnitudes (δ235) were 23.6 ± 12.5‰ and 370.4 ± 103.9‰, respectively. These results open up new perspectives on the re-enrichment of DU tailings, offering a potential biological alternative to obtain reprocessed natural-equivalent uranium. Additionally, the findings present implications for identifying biological signatures in the geologic records.

Anthropogenic extreme environments are emphasized as interesting sites for the study of evolutionary pathways, biodiversity, and extremophile bioprospection. Organisms that grow under these conditions are usually regarded as extremophiles; however, the extreme novelty of these environments may have favor adaptive radiations of facultative extremophiles. At the Iberian Peninsula, uranium mining operations have rendered highly polluted extreme environments in multiple locations. In this study, we examined the phytoplankton diversity, community structure, and possible determining factors in separate uranium mining-impacted waters. Some of these human-induced extreme environments may be able to sustain indigenous facultative extremophile phytoplankton species, as well as alleged obligate extremophiles. Therefore, we investigated the adaptation capacity of three laboratory strains, two Chlamydomonas reinhardtii and a Dictyosphaerium chlorelloides, to uranium-polluted waters. The biodiversity among the sampled waters was very low, and despite presenting unique taxonomic records, ecological patterns can be identified. The microalgae adaptation experiments indicated a gradient of ecological novelty and different phenomena of adaptation, from acclimation in some waters to non-adaptation in the harshest anthropogenic environment. Certainly, phytoplankton extremophiles might have been often overlooked, and the ability to flourish in extreme environments might be a functional feature in some neutrophilic species. Evolutionary biology and microbial biodiversity can benefit the study of recently evolved systems such as uranium-polluted waters. Moreover, anthropogenic extremophiles can be harnessed for industrial applications.

The extraction and processing of uranium (U) have polluted large areas worldwide, rendering anthropogenic extreme environments inhospitable to most species. Noticeably, these sites are of great interest for taxonomical and applied bioprospection of extremotolerant species successfully adapted to U tailings contamination. As an example, in this work we have studied a microalgae species that inhabits extreme U tailings ponds at the Saelices mining site (Salamanca, Spain), characterized as acidic (pH between 3 and 4), radioactive (around 4 μSv h−1) and contaminated with metals, mainly U (from 25 to 48 mg L−1) and zinc (from 17 to 87 mg L−1). After isolation of the extremotolerant ChlSP strain, morphological characterization and internal transcribed spacer (ITS)-5.8S gene sequences placed it in the Chlamydomonadaceae, but BLAST analyses identity values, against the nucleotide datasets at the NCBI database, were very low (

The ecological factors driving the occurrence of harmful algal blooms (HAB) have been traditionally analysed from a hydrological point of view, whereas the interplay between atmospheric oscillations and HAB has been scarcely explored. Here we address the possible link between atmospheric oscillations in SW Europe, using as proxies the North Atlantic Oscillation (NAO) and the Arctic Oscillation (AO) indices and the interannual variability in HAB. The yearly series (1973–2005) of mortality of water birds and fish in Donana National Park (SW Spain) due to toxins associated with the cyanobacterium Microcystis aeruginosa, and the monthly incidence (January 1999 to December 2007) of the paralytic shellfish poisoning (PSP) toxin produced by the dinoflagellate Gymnodinium catenatum in the Rias Baixas (NW Spain), were selected as models of HAB in SW Europe. The incidences of both toxic algal events were fitted to a binary logistic regression as a function of the atmospheric oscillation indices (with different delays in accordance with each toxic event) and a favourability function was then computed. The favourability for the wildlife mortality in Donana National Park was as a function of the AO averaged for the summer period (June–August) whereas the favourability for the incidence of PSP in the Rias Baixas was a function of the NAO recorded 1 month before the event. Since HAB have a relevant impact on ecosystems and human health, there is great interest in deciphering the ecological conditions favouring these events. Here we show that the atmospheric oscillations could be drivers of ecological processes linked to the occurrence of HAB in SW Europe. Moreover, the favourability functions relating NAO and AO indices with algal toxic events in SW Europe could be used as a powerful tool to predict toxic events.

English. The cyanobacterium Microcystis aeruginosa (Kützing) Kützing is notorious for forming extensive and toxic blooms but the genetic structure of natural populations, and in particular during blooms, remains to be explored. In order to add more knowledge about the genetic structure of M. aeruginosa, we compared phenotypic and genetic variabilities in seventeen strains of M. aeruginosa isolated from three adjacent water bodies in SW Spain (the consecutive reservoirs La Minilla and El Gergal, and small pools in Doñana National Park). For this purpose, a phenetic tree, based on six phenotypic traits (including morphology of cells, photosynthetic and respiratory performance, and toxin production) was compared with a phylogenetic tree built by using the 16S-23S rDNA Internal Transcribed Spacer sequence. Whereas the strains isolated from La Minilla showed a relatively homogeneous phenotype, the phenotypic traits of the remaining strains did not discriminate between the other two water bodies. On the other hand, only a strain isolated in Doñana National Park showed a slightly different ITS sequence, but the sequences from the remaining strains were similar. Possible explanations for this discrepancy between phenotypic and genetic diversities are discussed.Español. La cianobacteria Microcystis aeruginosa (Kützing) Kützing puede dar lugar a extensas floraciones tóxicas pero la estructura genética de las poblaciones naturales, y en particular durante las floraciones, ha sido escasamente estudiada. Con el objeto de profundizar en la estructura genética de M. aeruginosa, se han comparado las variabilidades fenotípica y genética de diecisiete cepas aisladas de tres cuerpos de agua adyacentes en el SW de España (los embalses consecutivos de La Minilla y El Gergal, y charcas en el Parque Nacional de Doñana). Para este fin, un árbol de similitud fenética basado en seis caracteres fenotípicos (que incluyen la morfología de las células, el rendimiento fotosintético y respiratorio y la producción de toxina) se comparó con un árbol filogenético construido mediante el uso de la secuencia de ITS 16S - 23S del ADNr. Las cepas aisladas en La Minilla mostraron un fenotipo relativamente homogéneo pero no se detectó un patrón de fenotipos que discriminase los otros dos cuerpos de agua. Por otro lado, sólo una cepa aislada en el Parque Nacional de Doñana mostró una secuencia ITS diferente, pero las secuencias de las cepas restantes fueron idénticas. Se discuten las posibles explicaciones para esta discrepancia entre la diversidad fenotípica y genética en M. aeruginosa.

Los Baños de Vilo (S Spain) is a natural spa characterized by extreme sulphureous waters; however, populations of chlorophyceans inhabit in the spa. The adaptation mechanisms allowing resistance by photosynthetic microorganisms to the extreme sulphureous waters were studied by using a modified Luria-Delbrück fluctuation analysis. For this purpose, the adaptation of the chlorophycean Dictyosphaerium chlorelloides and the cyanobacterium Microcystis aeruginosa (both isolated from non-sulphureous water) were analysed in order to distinguish between physiological adaptation (acclimation) and genetic adaptation by the selection of rare spontaneous mutations. Acclimation to the extreme water was achieved by D. chlorelloides; however, M. aeruginosa cells proliferated as a consequence of selection of favoured mutants (i.e. genetic adaptation). The resistant cells of M. aeruginosa appeared with a frequency of 7.1 × 10(-7) per cell per generation, and the frequency of the resistant allele, under non-selective conditions, was estimated to be 1.1 × 10(-6) per cells as a consequence of the balance mutation-selection. It could be hypothesized that the populations of eukaryotic algae living in the Los Baños de Vilo could be the descendants of chlorophyceans that arrived fortuitously at the spa in the past. On the other hand, cyanobacteria could quickly adapt by the selection of favoured mutants. The single mutation that allows resistance to sulphureous water from Baños de Vilo in M. aeruginosa represents a phenotypic burden impairing growth rate and photosynthetic performance. The resistant-variant cells of M. aeruginosa showed a lower acclimated growth rate and a decreased maximum quantum yield and photosynthetic efficiency, in comparison to the wild-type cells.

Marine ecosystems are affected by introduced species including microalgae. We propose that biofouling on ships’ hulls is a potentially important mechanism for microalgae dispersal worldwide. Biofouling samples, for phytoplankton composition analysis, were collected in Spanish Mediterranean ports from the hulls of ships that had completed oceanic journeys from other Mediterranean ports, and long journeys from the Atlantic and Indian Oceans. Samples representing the local population of phytoplankton either in the water column or attached to the biofouling of locally-based ship-hulls were used as controls. A broad variety of microalgae species (including toxic dinoflagellates), which were not present in the local phytoplankton populations were found on the biofouling film of the ships that had been on distant journeys. In spite of the presence of the antifouling paints containing toxic compounds, microalgae were able to rapidly adapt to these non-favourable conditions. Consequently, our study shows that ships’ biofouling seems to be a powerful vector for microalgae dispersal at a global scale due to the capacity of microalgae to attach to the biofouling film and to cope by adaptation mechanisms with antifouling compounds., This work has been financially supported by the Spanish Ministry of Sciences and Innovation through the grants CTM 2008-05680 C02-02 and CGL 2008-00652/BOS. Authors thank the financial support given by the Spanish Ministry of Education and Science, through FPU program.

The cyanobacterium Microcystis aeruginosa forms blooms that can consist of colonies. We have investigated how M. aeruginosa acclimatizes to changing light conditions such as can occur during blooms. Three different strains were exposed to two irradiance levels: lower (LL) and higher (HL) than the irradiance-onset saturation parameter. We measured the photosynthetic pigment concentrations, PSII photochemical efficiency, electron transport rate (ETR), irradiance-saturated ETR and ETR efficiency. The relationship between ETR and photosynthetic oxygen production and the excess in PSII capacity were also studied for one strain. Higher values of chlorophyll a and phycocyanin and lower values of total carotenoids were found under LL conditions in the three strains. The strains showed clear differences in the irradiance-saturated ETR and in ETR efficiency under both LL and HL treatments. No differences were found in the linear relationship between ETR and photosynthetic oxygen production under both irradiance treatments. LL-acclimated cells showed higher PSII excess capacity than HL ones, possibly because their higher pigment content could result in a higher light stress than HL cells when forming surface blooms. The fact that the genetically different strains show different photosynthetic physiologies suggests that the very dynamic light climate observed in lakes may allow their coexistence.

The roles of adaptation, chance, and history on evolution of the toxic dinoflagellate Alexandrium minutum Halim, under selective conditions simulating global change, have been addressed. Two toxic strains (AL1V and AL2V), previously acclimated for two years at pH 8.0 and 20°C, were transferred to selective conditions: pH 7.5 to simulate acidification and 25°C. Cultures under selective conditions were propagated until growth rate and toxin cell quota achieved an invariant mean value at 720 days (ca. 250 and ca. 180 generations for strains AL1V and AL2V, respectively). Historical contingencies strongly constrained the evolution of growth rate and toxin cell quota, but the forces involved in the evolution were not the same for both traits. Growth rate was 1.5–1.6 times higher than the one measured in ancestral conditions. Genetic adaptation explained two-thirds of total adaptation while one-third was a consequence of physiological adaptation. On the other hand, the evolution of toxin cell quota showed a pattern attributable to neutral mutations because the final variances were significantly higher than those measured at the start of the experiment. It has been hypothesized that harmful algal blooms will increase under the future scenario of global change. Although this study might be considered an oversimplification of the reality, it can be hypothesized that toxic blooms will increase but no predictions can be advanced about toxicity.

El control de la contaminacion necesita, hoy en dia, de sistemas de deteccion y analisis que permitan alcanzar altos niveles de especificidad y sensibilidad, con el fin de ser capaces de detectar la presencia de contaminantes cada vez mas diversos en cuanto a sus caracteristicas fisico-quimicas y que estan presentes en concentraciones cada vez mas bajas. Las microalgas son organismos fotosinteticos muy sensibles a los pequenos cambios que puedan producirse en el ambiente que los rodea, lo que los convierte en una herramienta muy util para la rapida deteccion (casi instantanea) de contaminantes presentes a niveles traza. Estos organismos microscopicos, que viven en los ecosistemas acuaticos, ofrecen una solucion versatil para la construccion de nuevos biosensores que demanda la actual normativa de calidad y seguridad medioambiental. Los biosensores microalgales que presentamos estan basados en la actuacion simultanea de dos genotipos uno sensible y otro resistente, obtenidos mediante un proceso de mejora genetica por seleccion artificial sin ser organismos geneticamente modificados. Estos biosensores especificos para cada uno de los contaminantes, permiten discriminar la presencia de un compuesto diana en un medio complejo, incrementando tambien la sensibilidad, algo no conseguido hasta el momento.

Lindane is especially worrisome because its persistence in aquatic ecosystems, tendency to bioaccumulation and toxicity. We studied the adaptation of freshwater cyanobacteria and microalgae to resist lindane using an experimental model to distinguish if lindane-resistant cells had their origin in random spontaneous pre-selective mutations (which occur prior to the lindane exposure), or if lindane-resistant cells arose by a mechanism of physiological acclimation during the exposure to the selective agent. Although further research is needed to determine the different mechanisms contributing to the bio-elimination of lindane, this study, however, provides an approach to the bioremediation abilities of the lindane-resistant cells. Wild type strains of the experimental organisms were exposed to increasing lindane levels to estimate lethal concentrations. Growth of wild-type cells was completely inhibited at 5mg/L concentration of lindane. However, after further incubation in lindane for several weeks, occasionally the growth of rare lindane-resistant cells was found. A fluctuation analysis demonstrated that lindane-resistant cells arise only by rare spontaneous mutations that occur randomly prior to exposure to lindane (lindane-resistance did not occur as a result of physiological mechanisms). The rate of mutation from lindane sensitivity to resistance was between 1.48 × 10(-5) and 2.35 × 10(-7) mutations per cell per generation. Lindane-resistant mutants exhibited a diminished fitness in the absence of lindane, but only these variants were able to grow at lindane concentrations higher than 5mg/L (until concentrations as high as 40 mg/L). Lindane-resistant mutants may be maintained in uncontaminated waters as the result of a balance between new resistant mutants arising from spontaneous mutation and resistant cells eliminated by natural selection waters via clone selection. The lindane-resistant cells were also used to test the potential of microalgae to remove lindane. Three concentrations (4, 15 and 40 mg/L) were chosen as a model. In these exposures the lindane-resistant cells showed a great capacity to remove lindane (until 99% lindane was eliminated). Apparently, bioremediation based on lindane-resistant cells could be a great opportunity for cleaning up of lindane- and other chlorinated organics-polluted habitats.

We tested if different adaptation strategies were linked to a stress gradient in phytoplankton cells. For this purpose, we studied the adaptation and acclimation of Dictyosphaerium chlorelloides (Naumann) Komarek et Perman (Chlorophyta) and Microcystis aeruginosa (Kutz.) Kutz. (Cyanobacteria) to different water samples (from extremely acid, metal-rich water to moderate stressful conditions) of the Agrio River-Caviahue Lake system (Neuquen, Argentina). Both experimental strains were isolated from pristine, slightly alkaline waters. To distinguish between physiological acclimation and genetic adaptation (an adaptive evolution event), a modified Luria-Delbruck fluctuation analysis was carried out with both species by using as selective agent sample waters from different points along the stress gradient. M. aeruginosa did not acclimate to any of the waters tested from different points along the stress gradient nor did D. chlorelloides to the two most acidic and metal-rich waters. However, D. chlorelloides proliferated by rapid genetic adaptation, as the consequence of a single mutation (5.4 × 10(-7) resistant mutants per cell per division) at one locus, in less extreme water and also by acclimation in the least extreme water. It is hypothesized that the stress gradient resulted in different strategies of adaptation in phytoplankton cells from nonextreme waters. Thus, very extreme conditions were lethal for both organisms, but as stressful conditions decreased, adaptation of D. chlorelloides cells was possible by the selection of resistant mutants, and in less extreme conditions, by acclimation.

Toxic blooms of the cyanobacterium Microcystis aeruginosa affect humans and animals in inland water systems worldwide, and it has been hypothesized that the development of these blooms will increase under the future scenario of global change, considering eutrophication and temperature increase as two important consequences. The importance of genetic adaptation, chance and history on evolution of growth rate, and toxin production of M. aeruginosa was studied under these new conditions. The experiment followed the idea of "replaying life's tape" by means of the simultaneous propagation of 15 independent isolates of three M. aeruginosa strains, which were grown under doubled nutrient concentration and temperature during c. 87 generations. Adaptation by new mutations that resulted in the enhancement of growth rate arose during propagation of derived cultures under the new environmental conditions was the main component of evolution; however, chance also contributed in a lesser extension to evolution of growth rate. Mutations were selected, displacing the wild-type ancestral genotypes. In contrast, the effect of selection on mutations affecting microcystin production was neutral. Chance and history were the pacemakers in evolution of toxin production. Although this study might be considered an oversimplification of the reality, it suggest that a future scenario of global change might lead to an increase in M. aeruginosa bloom frequency, but no predictions about the frequency of toxicity can be made.

In order to study adaptation of microalgae to petroleum contamination, we have examined an environmental stress gradient by crude oil contamination in the Arroyo Minero River (AMR), Argentina. Underground crude oil has constantly leaked out since 1915 as a consequence of test drilling for possible petroleum exploitation. Numerous microalgae species proliferated in AMR upstream of the crude oil spill. In contrast, only four microalgal species were detected in the crude oil spill area. Species richness increases again downstream. Microalgae biomass in the crude oil spill area is dominated by a mesophile species, Scenedesmus sp. Effects of oil samples from AMR spill on photosynthetic performance and growth were studied using laboratory cultures of two Scenedesmus sp. strains. One strain (Se-co) was isolated from the crude oil spill area. The other strain (Se-pr) was isolated from a pristine area without petroleum contamination. Crude oil has undetectable effects on Se-co strain. In contrast crude oil rapidly destroys Se-pr strain. However, Se-pr strain can adapt to low doses of petroleum (≤ 3% v/v total hydrocarbons/water) by means of physiological acclimatization. In contrast, only rare crude oil-resistant mutants are able to grow under high levels of crude oil (≥ 10% v/v total hydrocarbons/water). These crude oil-resistant mutants have arisen through rare spontaneous mutations that occur prior to crude oil exposure. Species richness in different areas of AMR is closely connected to the kind of mechanism (genetic adaptation vs. physiological acclimatization) that allows adaptation. Resistant-mutants are enough to assure the survival of microalgal species under catastrophic crude oil spill.

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 Fv/Fm, ETRmax and α estimations) was observed after only 1 h 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 2.76 × 10−5 oil-resistant mutants per cell division. Apparently, rare spontaneous preselective mutations are able to assure the survival of microalgae in oil-polluted environments.

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–Delbruck 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 × 10−6 mutants per cell per generation in both strains of S. intermedius, and of 9.2 × 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 × 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.

SUMMARY Four species of eukaryotic algae proliferate in the sul- fureous, 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- Delbruck 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. Pre- selective, 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 photosyn- thesizers 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.

At least six species of eukaryotic microalgae inhabit the acidic (pH 2.4–2.7), metal-rich mine waters from ponds in the copper mine district of Mynydd Parys (N Wales, UK). Consequently, these ponds constitute interesting natural laboratories for analysis of adaptation by microalgae to extremely stressful conditions. To distinguish between the pre-selective and post-selective origin of adaptation processes that allow the existence of microalgae in these ponds, a Luria-Delbruck fluctuation analysis was performed with the chlorophycean Dictyosphaerium chlorelloides isolated from non-acidic waters. In this analysis, natural Mynydd Parys pond water (MPW) was used as selective factor. Pre-selective, resistant D. chlorelloides cells appeared with a frequency of 1.6 × 10−6 per cell per generation. MPW-resistant mutants, with a diminished Malthusian fitness, are maintained in non-extreme waters as the result of a balance between new MPW-resistant cells arising by mutation and MPW-resistant mutants eliminated by natural selection (equilibrium at ca. 19 MPW-resistant per 107 wild-type cells). We propose that the microalgae inhabiting these stressful ponds could be the descendents of chance mutants that arrived in the past or are even arriving at the present.

Several species of microalgae, closely related to mesophilic lineages, inhabit the extreme environment (pH 2.5, high levels of metals) of the Spain’s 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–Delbruck 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 × 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.

Evolutionary changes depend on adaptive evolution and two other components that traditionally have not been taken into account: chance and history. However, concepts in evolutionary biology are very often supported by descriptive studies but not by empirical approach. In order to disentangle the three components of evolutionary change, we have investigated the effects of long-term selection pressure on fitness and cell size in the bloom-forming dinoflagellate, Prorocentrum triestinum Schiller, under increased nutrient concentrations and temperature. Two strains of P. triestinum, previously adapted to grow under standard conditions (f/2 medium, 20 degrees C), were grown under increased nutrient concentrations and temperature (f medium, 25 degrees C) until fitness and cell size were invariant as a function of time (720 days, corresponding to c. 400 generations). The mean value for fitness of both strains of P. triestinum showed a significant increase whereas a decrease in mean cell size was found. The changes in fitness have adaptive value, but the changes in cell size were due to both adaptation and the contribution of chance-plus-history.

The adaptation mechanisms of microalgae to grow in contaminated waters were analysed using a chlorophyta species under formaldehyde exposure as experimental model. Cultures initially collapsed after exposure to 16 ppm formaldehyde, but occasionally resistant cells were able to grow after further incubation. Resistant cells arose by rare spontaneous mutations that appeared before the exposure to formaldehyde (mutation rate=3.61 x 10(-6)), and not as result of physiological mechanisms. Although mutations may be the mechanisms that should allow the survival of microalgae in polluted waters in a world under rapid global change, mutants have a diminished growth rate.

In the last decades, relevant efforts have been made to reduce the cancer incidence in the European Union. The prevention programmes against cancer have obtained satisfactory results except for colorectal cancer (CRC). Identification of risk factors is primordial to plan preventive strategies for CRC. We hypothesize that shellfish consumption is increasing CRC incidence. DSP toxins, present in some seafood products, seem to behave like tumour agents. There are no relevant studies on real health-risk of consuming DSP toxins, just some experimental and ecological evidence. Preventive interventions for reducing CRC risk must be approached through the collaboration of governmental, health and environmental sectors as a single regulatory agency. Sometimes, shellfish accumulates diarrhetic shellfish poisoning (DSP) toxins (i.e. okadaic acid and its derivatives) which provoke a gastrointestinal illness (DSP syndrome). Furthermore, DSP toxins are tumour promoters that could increase CRC risk. The current regulation about level of DSP toxins in shellfish meat is only centred on reduction of the gastrointestinal symptoms. Unfortunately, legal levels of DSP toxins in shellfish are enough to increase CRC risk. A review of legislation on DSP toxins is urgent.

We tested if different adaptation strategies were linked to a stress gradient in phytoplankton cells. For this purpose, we studied the adaptation and acclimation of Dictyosphaerium chlorelloides (Naumann) Komárek et Perman (Chlorophyta) and Microcystis aeruginosa (Kütz.) Kütz. (Cyanobacteria) to different water samples (from extremely acid, metal-rich water to moderate stressful conditions) of the Agrio River-Caviahue Lake system (Neuquén, Argentina). Both experimental strains were isolated from pristine, slightly alkaline waters. To distinguish between physiological acclimation and genetic adaptation (an adaptive evolution event), a modified Luria-Delbrück fluctuation analysis was carried out with both species by using as selective agent sample waters from different points along the stress gradient. M. aeruginosa did not acclimate to any of the waters tested from different points along the stress gradient nor did D. chlorelloides to the two most acidic and metal-rich waters. However, D. chlorelloides proliferated by rapid genetic adaptation, as the consequence of a single mutation (5.4 × 10(-7) resistant mutants per cell per division) at one locus, in less extreme water and also by acclimation in the least extreme water. It is hypothesized that the stress gradient resulted in different strategies of adaptation in phytoplankton cells from nonextreme waters. Thus, very extreme conditions were lethal for both organisms, but as stressful conditions decreased, adaptation of D. chlorelloides cells was possible by the selection of resistant mutants, and in less extreme conditions, by acclimation.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., In the current scenario of global change, the impact of anthropogenic stressors is affecting the aquatic ecosystems, especially essential microorganisms such as phytoplankton, driving toward a biodiversity crisis. Classic ecotoxicology studies, focused on the immediate tolerance to pollution, have provide an over simplistic understanding of the long time impact of pollutants on phytoplankton (because of the usual misconception that evolutionary changes can only take place at long-term), inadequate to enable a suitable environmental risk assessment (ERA). Currently, concepts such as predictive ecology and integrating strategies are rising rapidly in prominence with regard to forecasting phytoplankton response to human impact. This review compiles the state of the art of multigenerational and evolutionary experimental studies and the mechanisms that trigger rapid adaptation in phytoplankton to anthropogenic stressors, highlighting the importance of ecology and evolution. Ecological realisms is one of the challenging parts of the stressors hazard, considering the broad phytoplankton diversity and the multifactorial character of the natural ambiances. Field and community experiments contribute to a better discerning of ecology interactions and network relations. Moreover, laboratory experiments exploring evolutionary mechanisms that allow rapid adaptation to contaminants (e.g., fluctuation analysis or ratchet procedures) have proven to be useful linking concentration of pollutants and adaptation strategy. When environmental change exceeds the range of variation that can be coped with by organisms through plasticity, selection processes may occur and evolutionary dynamics take place. Ecology and evolution are necessary to enhance the ERA knowledge and novel experiments may well emerge from the when contemplated as whole., Supported by the Spanish Secretaría de Estado de Investigación, Desarrollo e Innovación (Grant CTM 2012-34757 and Grant CTM 2013-44366-R) and Junta de Andalucía project RNM-04744.

Although populations of cyanobacteria are usually considered to be clonal, their capacity to survive environmental changes suggests intrapopulation genetic variation. We therefore estimated the genetic variability on the basis of two processes important for any photoautotroph - photochemical and nonphotochemical quenching - as well as photosynthetic pigment concentrations. For this purpose, two parameters related to photochemical and nonphotochemical quenching were measured using specific experimental and statistical procedures, in 25 strains of the cyanobacterium Microcystis aeruginosa, along with their contents of chlorophyll a, total carotenoids and phycocyanin. The experimental procedure allowed discrimination between genetic and nongenetic (or residual) variability among strains. The high genetic variability found in photosynthetic pigments and both photosynthetic parameters denotes large differences even among strains isolated from the same community. The high genetic diversity within a population could be important for the evolutionary success of cyanobacteria.

Adaptation of Microcystis aeruginosa (Cyanobacteria) to resist the herbicide glyphosate was analysed by using an experimental model. Growth of wild-type, glyphosate-sensitive (Gs) cells was inhibited when they were cultured with 120 ppm glyphosate, but after further incubation for several weeks, occasionally the growth of rare cells resistant (Gr) to the herbicide was found. A fluctuation analysis was carried out to distinguish between resistant cells arising from rare spontaneous mutations and resistant cells arising from other mechanisms of adaptation. Resistant cells arose by rare spontaneous mutations prior to the addition of glyphosate, with a rate ranging from 3.1 × 10−7 to 3.6 × 10−7 mutants per cell per generation in two strains of M. aeruginosa; the frequency of the Gr allele ranged from 6.14 × 10−4 to 6.54 × 10−4. The Gr mutants are slightly elliptical in outline, whereas the Gs cells are spherical. Since Gr mutants have a diminished growth rate, they may be maintained in uncontaminated waters as the result of a balance between new resistants arising from spontaneous mutation and resistants eliminated by natural selection. Thus, rare spontaneous pre-selective mutations may allow the survival of M. aeruginosa in glyphosate-polluted waters via Gr clone selection.

Inter-strain variability in pH compensation point (pH c ) in the cyanobacterium Microcystis aeruginosa has been investigated. The pH c allows one to discriminate whether the organism is able to take up HCO 3 − as inorganic carbon (C i ) source in photosynthesis. Eight subgroups were found according to the pH c value, ranging from 10.44 ± 0.22 to 11.67 ± 0.05. The high variability in pH c (and consequently, in the capacity to use HCO 3 − as C i source) suggested that different HCO 3 − use mechanisms could occur in M. aeruginosa and, from an evolutionary point of view, this trait is not under high natural selective pressure.

Traditionally, morphometrical traits are used to classify cyanobacteria. However, neither the importance of genetic factors in morphological variability nor genetic correlations between different morphometrical traits are known. Six morphometrical traits of individual cells and six morphometrical traits of colonies have been measured in 21 strains of Microcystis aeruginosa. A fixed model and a mixed model allowing genetic relationships between traits were both used to estimate genetic and residual correlations, and genetic variance/total phenotypic variance ratios were used to estimate the 12 morphometrical traits. Results showed that (1) colony size has a low genetic correlation with traits of individual cells; (2) traits measured on individual cells present a higher genetic variance/total phenotypic variance ratio than the corresponding trait in colonies; and (3) genetic correlations between directly measured traits (maximal diameter and axis, minimal diameter and axis, perimeter, and surface) ...

Microcystis aeruginosa (Cyanobacteria) is known to be the main source of toxic blooms affecting humans and animals in inland water systems. Usually, this toxic species occurs in dense blooms with p...

The existence of Giffen behaviour (i.e. the opposite situation to the Law of Demand in which consumers respond to a rising price of a certain good by demanding more of it) associated with poor consumers is one of the major controversies in economics. However, economists rarely consider that nothing makes sense except in light of evolution. In this paper, we prove the existence of Giffen behaviour in animals that exhibit no intelligent reasoning. Sardines feed on phytoplankton, and zooplankton. If the amount of phytoplankton is greater than zooplankton, then Sardines fit the Law of Demand, but if the amount of phytoplankton is smaller, the Sardines apparently show Giffen behaviour. Evolutionary population genetics models show that Giffen behaviour has more adaptive value than follow the Law of Demand under resource scarcity. Since overwhelming animal species live in poverty, Giffen behaviour may be the common economic strategy on the Earth.

The increasing rates of global extinction due to human activities necessitate studies of the ability of organisms to adapt to the new environmental conditions resulting from human disturbances. We investigated the evolutionary adaptation of a microalga to sudden environmental change resulting from exposure to novel toxic chemical residues. A laboratory strain of Dictyosphaerium chlorelloides (Naum.) Kom. and Perm. (Chlorophyceae) was exposed to increasing concentrations of the modern contaminant 2,4,6-trinitrotoluene (TNT). When algal cultures were exposed to 30 mg.L - 1 TNT, massive lysis of microalgal cells was observed. The key to understanding the evolution of microalgae in such a contaminated environment is to characterize the TNT-resistant variants that appear after the massive lysis of the TNT-sensitive cells. A fluctuation analysis demonstrated unequivocally that TNT did not facilitate the appearance of TNT-resistant cells; rather it was found that TNT-resistant cells appeared spontaneously by rare mutations under nonselective conditions, before exposure to TNT. The estimated mutation rate was 1.4 X 10 - 5 mutants per cell division. Isolated resistant mutants exhibited a diminished fitness in the absence of TNT. Moreover, the gross photosynthetic rate of TNT-resistant mutants was significantly lower than that of wild-type cells. Competition experiments between resistant mutants and wild-type cells showed that in small populations, the resistant mutants were driven to extinction. The balance between mutation rate and the rate of selective elimination determines the occurrence of about 36 TNT-resistant mutants per million cells in each generation. These scarce resistant mutants are the guarantee of potential for adaptation.

The toxic spill of acid wastes rich in heavy metals/metalloids (AWHM) from the Aznalcollar mine in April 1998, threatening the Donana National Park, is considered to be the worst environmental disaster related to acute pollution in Spanish history. The aim of this work was to study the adaptation of microalgae (which play an important role as primary producers) from AWHM sensitivity to AWHM resistance by using the alga Scenedesmus intermedius as an experimental model. The Malthusian parameter (m) and the carrying capacity (K) were reduced by mud and soil samples collected from the affected area. A dose–effect analysis showed that fitness progressively diminished with increasing sample concentration. A fluctuation analysis demonstrated that AWHM-resistant cells arose by rare spontaneous mutations that occurred randomly prior to the incorporation of the AWHM. The rate of spontaneous mutation from AWHM sensitivity to AWHM resistance was 2 12x10−O mutants per cell division. A competition experiment between wild-type AWHM-sensitive cells and AWHM-resistant mutants showed that in small populations the AWHM-resistant mutants are driven to extinction in the absence of selection for AWHM resistance. The resistant phenotypes are maintained in the absence of AWHM as the result of a balance between spontaneous mutation and natural selection, so that about 43 AWHM-resistant mutants per million cells are present in the absence of AWHM. Our experimental model suggests that mutation is essential for adaptation of microalgal populations to environmental changes. Rare spontaneous pre-adaptive mutation is enough to ensure the survival of microalgal populations in contaminated environments when the population size is large enough.

Cyanobacteria are a source of potent toxins among which the microcystin (a hepatotoxic peptide encoded by the mcy gene cluster of Microcystis spp. ) is a frequent cause of poisoning in inland waters worldwide. Although the molecular basis of microcystin production is known, its role is still unknown. It was suggested that microcystin production have a metabolic cost that could be offset by some benefit (e.g. protection from grazing). We check that: i) microcystin-producing and non-producing strains occurs simultaneously in the Microcystis spp. blooms, ii) evolutionary forces (mutation, genetic drift) control frequencies of microcystin production and non-production strains, and iii) microcystin producing strains have diminished fitness compared with non-producing strains. We employ evolutionary game theory to explain the maintaining of microcystin-producing genotypes in natural populations of Microcystis spp . A two-strategy (to produce or not microcystin), two-players game of cooperators (microcystin-producing genotypes) and cheaters (non-producing genotypes) explains the coexistence of both genotypes in the same bloom. A bloom composed mostly by the microcystin-producing “cooperators” genotype, the “temptation of defection” (increase of non-producing genotypes) is counteracted by kin selection, which enable that natural selection can favour the cooperators. The closest related individuals occur within cyanobacteria blooms, cyanobacteria reproduces asexually providing sets of clones.

Copper is one of the most frequently used algaecides to control blooms of toxic cyanobacteria in water supply reservoirs. Among the negative impacts derived from the use of this substance is the increasing resistance of cyanobacteria to copper toxicity, as well as changes in the community structure of native phytoplankton. Here, we used the ratchet protocol to investigate the differential evolution and maximum adaptation capacity of selected freshwater phytoplankton species to the exposure of increasing doses of copper. Initially, a dose of 2.5 μM CuSO4·5H2O was able to completely inhibit growth in three strains of the toxic cyanobacterium Microcystis aeruginosa, whereas growth of the chlorophyceans Dictyosphaerium chlorelloides and Desmodesmus intermedius (represented by two different strains) was completely abolished at 12 μM. A significant increase in resistance was achieved in all derived populations during the ratchet experiment. All the chlorophyceans were able to adapt to up to 270 μM of copper sulfate, but 10 μM was the highest concentration that M. aeruginosa strains were able to cope with, although one of the replicates adapted to 30 μM. The recurrent use and increasing doses of copper in water reservoirs could lead to the selection of copper-resistant mutants of both chlorophyceans and cyanobacteria. However, under high concentrations of copper, the composition of phytoplankton community could undergo a drastic change with cyanobacteria being replaced by copper-resistant chlorophyceans. This result stems from a distinct evolutionary potential of these species to adapt to this substance. © 2014 Springer-Verlag Berlin Heidelberg., This work has been financially supported by the Spanish Ministry of Science and Innovation through the Grants CTM2008-05680 C02-01/MAR, CGL2008-00652/BOS and by Junta de Andalucía Research Group RNM-115.

Residues contribute to water pollution and pose a challenge to microalgal populations because numerous contaminants are toxic to microalgae, even in the micromolar range. Adaptation of microalgae from herbicide sensitivity to herbicide resistance was analysed by an ecological genetic approach, using the unicellular alga Dictyosphaerium chlorelloides (Chlorophyceae) as an experimental model. A dose–effect study showed that the Malthusian parameter under conditions of r selection in an uncrowded environment and the carrying capacity under conditions of K selection in an crowded environment were both restricted even by low concentrations (< 1 μM) of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) herbicide. When a culture was treated with 50 μM DCMU, it cleared after a few days, as a result of destruction of sensitive cells by the herbicide. However, after further incubation for several days, the culture sometimes regained colour, owing to the growth of cell variants resistant to the herbicide. A fl...

Antibodies were developed against cell surface antigens of Prorocentrum lima (Ehrenberg) Dodge, P. triestinum, P. micans Ehrenberg, P. minimum (Pavillard) Schiller and P. rostratum Stein. Positive antigenic reactions were visualized with epifluorescence microscopy, using a fluorescein isothiocyanate (FITC) indirect immunofluorescence assay. An immunoassay with a secondary magnetic bead-conjugated antibody was also used. Polyclonal antisera against P. lima and against P. rostratum were species-specific. In contrast, the other antisera showed weak cross-reactions. The antisera with insufficient specificity were improved by exposure to cross-reacting antigens. Thus, species-specific preadsorbed antisera were obtained against each species. A set of strain-specific antibodies against P. lima clones were also prepared by preadsorption. The immunomagnetic bead assay also allowed us to separate P. lima cells from sediment surface samples or macroalgae. Antibody binding patterns were used to establish phenetic relationships among species/clones examined. P. micans, P. triestinum and P. minimum appeared as closely related. In contrast, P. lima, and P. rostratum emerged distant from the other Prorocentrum species. Additionally, serological differences between natural populations of P. lima (a DSP toxin-producing benthic dinoflagellate) were detected.