acesap

Activity 2: Adaptation - Aquatic Environments

A2-wp1 - Genetic diversity and adaptive traits in the filamentous cyanobacterium Planktothrix rubescens

Participants: Domenico D'Alelio, Andrea Gandolfi, Monica Tolotti, Adriano Boscaini, Giovanna Flaim, Graziano Guella, Nico Salmaso

Planktothrix rubescens (DeCandolle ex Gomont) Anagnostidis et Komárek 1988 (Cyanoprokaryota; Order: Oscillatoriales; Family: Phormidiaceae; Subfamily: Phormidioideae) is a freshwater microcystins-producing filamentous cyanobacterium living in the planktonic environment, in both meso-eutrophic and strongly thermally stratifying large lakes and stagnant waters, where it produces red water blooms during the summer season; in winter, it often colours the water red under the ice. The species occurs in several regions in northern temperate zones, where it effectively colonizes limnetic basins (lakes in Austria; SE France, Germany, Switzerland, N Italy etc.); outside of these limited areas, the species occurs occasionally over the whole temperate zone. Seasonal blooms of the species were reported in sub-Alpine lakes in Northern Italy (i.e. Como, Garda, Idro, Ledro, Levico, Pusiano). In this organism, cells are joined into straight or slightly curved thricomes, solitary, free-floating, which sometimes form purple-red (or, when dried, violet) irregular clusters that, in turns, accumulate in dense blooms provoking water discolorations.

In the frame of the project ACE-SAP, natural populations of Planktothrix rubescens will be collected from a set of key lakes in Trentino, distributed along morphometric (with focus on maximum depth), altitudinal and trophic gradient, where water temperature, light and chemical dynamics will be investigated in detail. Taxonomical identification of the species used in this study will be carried out with classical morphometric methods (light microscopy). Environmental samples from different habitats (different lakes and different depths from each lake) will be used for the isolation of single filaments used as inoculum for cultures in controlled experimental conditions. Both strains corresponding to environmental samples and pure cultures will be characterized metabolically. Analysis of the secondary metabolites and membrane lipid profiles will be carried out through advanced mass spectrometric techniques (MALDI/TOF and LC-ESI-ION TRAP) and, eventually, through Nuclear Magnetic Resonance measurements if the investigated strains will produce new metabolites which require a full and ab initio structural elucidation. Genetic analysis will include specifically the characterization of genes involved in the production of gas vesicles (gvp genes). Upon identification of temperature and light optima for different strains composing natural populations of Planktothrix, quality and quantity of target secondary metabolites will be investigated in cultures under optimal and stress (temperature, light) conditions. The genetic characterization of strains producing different types of gas vesicles will be carried out from cultured natural strains.

To date, sampling sessions have been carried out in the Levico Lake, where the planktonic community was collected from the water column by using a phytoplankton net (mesh = 40 µm). Vertical net sampling - i.e. from the bottom of the lake (ca. 40 m) to the surface - allowed the collection of the actual planktonic community, which was successively sorted out in the lab. Though the species was not producing a dense bloom in the lake waters at the time of the sampling, several strains of P. rubescens were identified and isolated in light microscopy and were eventually brought in culture. These cultures are now incubated under costant conditions (temperature = 13°C, photoperiod = 12:12h L:D, irradiance < 40 µmol photon · m-2 · s-1) in order to get material enough to begin preliminary biomolecular analysis, which are aimed at i) veryfing the usefulness of existing protocols and ii) standardising methods to be used during the species' bloom season, for project's purposes.

A2-wp2 - Target species: Bangia atropurpurea (Roth) C. Agardh

Participants: Marco Cantonati, Daniel Spitale, Alessia Scalfi, Nicola Angeli, Graziano Guella, Rita Frassanito, Carlo Andreoli, Nicoletta La Rocca, Isabella Moro, Katia Sciuto

Bangia atropurpurea is widely distributed in marine intertidal areas as well as in some freshwater habitats (rivers and lakes in North America, Europe and Asia). Concerning the distribution in Lake Garda, B. atropurpurea occurs mainly on rocky substrates in the central-northern Garda and sporadically in the southern part. B. atropurpurea typically occurs in the upper and mid eulittoral zones. Here it experiences, both diurnally and seasonally, extreme environmental fluctuations, resulting in desiccation, freezing, osmotic and radiation stress (including UV). Since L. Garda experiences wide seasonal water-level fluctuations, Bangia is supposed to be adapted to a very stressful habitat. Understanding the mechanisms of resistance to UV exposition and desiccation of B. atropurpurea is the main aim of our study within the ACE-SAP Project.

When growing outside the water, B. atropurpurea exhibits a set of ecological, phenological, and bioorganic adaptations. Preliminary results suggest that populations of B. atropurpurea grow desynchronized along a small-scale spatial gradient outside the water. As a consequence, near the water mainly wider filaments (monosporangia) occur while the upper zone is filled mainly by vegetative filaments. The patterns observed suggest that B. atropurpurea might possess different levels of tolerance according to the phenological state. In addition, along the same gradient, B. atropurpurea shows several shifts of pigments concentration, probably related to the different duration of air exposure. These preliminary results suggest that the combination of adaptive traits in B. atropurpurea changes along the depth gradient.

Since the beginning of 2008, the seasonal cycle of B. atropurpurea has been studied by means of frequent (fortnightly, monthly) sampling surveys. Detailed seasonal studies are being carried out in two sites (eastern shore, northern part of the lake). Moreover, the distribution of the target species in the whole lake has been investigated considering six localities, and, within each one, an impacted and a non-impacted site + replicates. Diatoms epiphytic on B. atropurpurea were studied in the two main sampling localities while epilithic diatom communities (used also for an ecological characterization of the shores) were investigated in the six localities. Results have already been (3rd Central European Diatom Meeting, Utrecht, The Netherlands; 57th North American Benthological Society Meeting, Grand Rapids, MI, USA) and will be (7th International Symposium "Use of Algae for Monitoring Rivers", Luxembourg) presented at international congresses.We are planning a field experiment in which artificial substrata (limestone tiles) previously colonized by B. atropurpurea are translocated to obtain two main treatments: (i) moisture level (hydrated and exposed to air) and (ii) radiation (ambient, ambient minus UVA, and ambient minus UVA and UVB). Within these main treatments, we will study the response of B. atropurpurea with ecological, morphological, target metabolites analysis, and genetic approaches. Candidate genes will be selected on the basis of literature and preliminary ecological, morphological, ultrastructural, and bioorganic analyses. The expression of the selected genes at the different experimental conditions will be assessed by RT-PCR and/or real time PCR. Stress induced by desiccation and by different radiation types will be evaluated by morphological and ultrastructural analysis, paying special attention to cellular membranes organization and to the stress-protection compounds. In addition, photosynthetic apparatus will be investigated concerning a set of specific target proteins (i.e. photosystem II reaction centre protein D1).

A2-wp3 - The adaptive potential of natural populations of two aquatic insect species in relation to temperature variations and pollutants

Participants: Paola Bernabò, Luigi Caputi, Alessandra Franceschini, Rita Frassanito, Graziano Guella, Olivier Jousson, Valeria Lencioni

Knowledge as to how aquatic insects will potentially react and adapt in face of increasing human impacts is one of the major challenge in prediction of future freshwater biodiversity trends.Two main drivers of biodiversity change have been individuated, global warming and land use. In relation to these drivers, two adaptive traits were selected, resistance to adverse temperature conditions and resistance to pollutants, in two target insect species (Diptera: Chironomidae): the cold stenothermal and stenotope Pseudodiamesa branickii (Nowicki, 1873) and the euriecious Chironomus riparius Meigen, 1804. The former is frequent in cold mountain springs and streams (<7-8 °C), the latter colonizes warm and eutrophic lakes and rivers and agricultural/industrial waste waters. The general aim of this WP is to give new insights to understand: 1) how could aquatic insects react to the oncoming temperature variation related to global climate change; 2) how aquatic insects from polluted freshwaters develop toxic resistance and detoxification capacity. This by the i) characterisation of genetic determinants of resistance to abiotic stresses in natural populations of the two selected target species and ii) definition of the role of metabolites such as proteins and sugars and of the membrane lipid composition in development of such resistances.

Five candidate genes have been indicated as determinants for resistance to cold and warm temperature (hsc70, hsp70, hsp90, afps) and to pollutants (cytP450). Heat shock proteins are known to be involved in temperature variation resistance in many organisms, as well as antifreeze proteins in cold resistance and cytochrome P450 monooxygenase in detoxification processes. Experiment will be carried out on IV instar larvae of P. branickii collected in the Noce Bianco stream (Trentino) and of C. riparius collected in the Lambro River (Lombardy) and reared in the laboratory. Survival curves will be performed on larvae exposed to different temperatures and toxic compounds. The expression levels of all genes will be analysed in stressed and control larvae via quantitative real-time RT-PCR. The main membrane lipid components (fatty acids, glycerolipids and phospholipids) will be characterized with mass spectrometry techniques. Particular attention will be paid to lysophospholipids and phospholipids, membrane constituents that are expected to change in response to freezing, both in the acyl chains and/or in their polar heads. The identification and quantification of cryoprotectans such as polyols and sugars involved in cold hardiness will be also carried out by different spectroscopic techniques. Overall, the experimental approach outlined will lead to a better understanding of the abiotic factors that represent major selective constrains to various aquatic taxa.

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