Temporal variability of Oscillatoria sp. in extreme environment: a sulfide-rich spring outflow

Time from the first sampling date abr ago dic abr Relative contribution to total Chl a (%) 0 20 40 60 80 100 Bluegreen algae Green algae Diatoms&dinoflagellates Cryptomonads Chlorophyll a (g/l) 0 2 4 6 8 10 12 Total chlorophyll a Sulfide (M) 050 100 150 200 250 Biovolume (m3/ml) 0.0 5.0e+7 1.0e+8 1.5e+8 2.0e+8 2.5e+8 3.0e+8 3.5e+8 Oscillatoria sp. (m3/ml) Abundance (cells/ml) 0 10 20 30 40 50 60 70 Oscillatoria sp. (cells/ml) Sulfide (M) 050 100 150 200 250 Bluegreen algae (g Chl a /ml) 0 2 4 6 8 10 12 Time from first sampling date abr ago dic abr Precipitation (mm) 0 20 40 60 80 100 120 140 160 180 Precipitation Sulfide (M) 0 50 100 150 200 250 Sulfide Temporal variability of the cyanobacterium Oscillatoria sp. in extreme environment -a sulfide-rich hot spring outflow Andreas Reul1, Elena Martín-Clemente2, Ignacio José Melero-Jiménez2, Miguel Hernández-López3, Francisco Javier Toro2, Elena BañaresEspaña 2, Antonio Flores-Moya2, María Jesús García-Sánchez2 1Facultad de CienciasUniversidad de Málaga, Departamento de Ecología y Geología, Málaga, Spain. 2Facultad de CienciasUniversidad de Málaga, Departamento de Biología Vegetal, Málaga, Spain. 3Facultad de CienciasUniversidad de Málaga, Departamento de Química anlítica, Málaga, Spain Los Baños de la Hedionda (Andalucía, southern Spain) is a sulfide-rich hot spring outflow (Fig. 1). While sulfide is appreciated in thermal bath, sulphide is toxic to most oxygenic photosynthetic organisms because it irreversibly blocks photosystem II. In spite of being a site of public use, and provide an interesting case to conditions for investigate on extremophile organisms, little is known about the temporal variability of the sulfide concentration and photosynthetic organisms inhabiting this extreme ecosystem. Here we present the first annual cycle of phytoplankton composition related to sulfide concentration. Introduction Material and methods Casares Jaén Sevilla Córdoba Huelva GranadaAlmería Spain France Algeria Morocco Italy Portugal Tunisia Switzerland Italy Germany Andorra Monaco Casares Cádiz Málaga Fig. 1 Location of the sulfide-rich hot spring outflow “The Hedionda”. Sampling site (a) and public use of the sulfide–rich outflow during summer (b). Ciliate (c) and Oscillatoria sp. ciliate aggregate (d) Sampling site (a) (b) (c) 100 um (d) 100 um Monthly and weekly field sampling was carried out between 11/03/2016 and 30/05/2017. At each sampling day, pH and sulfide concentration were measured in situ with a (Hanna HI 9125 and DR900 Multiparameter Portable Colorimeter respectively). Then, 5L samples were taken in the morning (9:00-11:00h), placed in polyethylene bottles and maintained in cold and dark during the 1h transport to the laboratory at the University of Málaga. At the laboratory, total chlorophyll a concentration and phytoplankton composition were measured with a submersible fluorospectrometer (BBE-Moldaenke FluoroProbe). The fluorospectrometer discriminated between the main phytoplanktonic groups (i.e. diatoms and dinoflagellates, cyanobacteria, green algae and cryptophytes) based on the relative fluorescence intensity of chlorophyll a (Chl a) at 680 nm, following sequential light excitation by 5 Light-Emitting Diodes (LEDs) emitting at 450, 525, 570, 590 and 610 nm [12]. For abundance and size estimation of Oscillatoria sp., 2L of sample were concentrated on a 45m mesh and recovered in 20ml. Then each sample was passed through the FlowCAM equipped with a 100 μm flow cell and a 100-fold magnification (10x objective). The analysis was performed in autoimage mode, where individual pictures of each particle in the vision field are taken. Afterwards phytoplankton abundance, size and biovolume were estimated by manual reprocessing of the original data fields [3]. Meteorological data were downloaded from the meteorological sampling station of Estepona [4]. Discussion Sulfide, chlorophyll a concentration and Oscillatoria sp. abundance and biovolumen show a clear annual cycle, with a hot, dry and sulphide-rich period (June-November), and a colder, sulfide-poor period (December-May). Low sulfide concentration is related to dilution by recharging of the aquifer in winter and spring. It is worth to note that highest Chl a concentration as well as highest Oscillatoria sp. abundance were found during the period where toxic sulfide concentration was highest. Low Chl a concentration during low sulfide concentration period could be due to higher cell loss of phytoplankton by water runoff as could be compensated by growth rate. However, algal loss by runoff would affect all planktonic groups, but the other taxa remain in similar concentration during the whole year (<1-2 mg Chl a /L). Which advantages explain the success of Oscillatoria sp.? Physiology: Growth rate as a function of sulfide concentration is shown in poster 656, by Martín-Clemente et al. (2017) and could explain increasing Oscillatoria sp. abundance in summer. Morphology: Taking into account that the other phytoplanktonic groups are not filamentous and small sized (<45 m), filamentous algae and aggregates (Oscillatoria sp.) might get trapped on other water plants and branches giving Oscillatoria sp. an advantage in a fluvial system. Trophic interactions: Apart of phytoplankton we also found some ciliates (Fig. 1c), specially in summer, which might control smaller sized phytoplankton. Images from fresh samples passing through the FlowCAM show Ciliate-Oscillatoria aggregates (Fig. 1d). Thus ciliates might also predate on Oscillatoria sp., however big-sized Oscillatoria sp. aggregates could prevent top down control until elevate ciliate abundance, which might occur under elevate sulfide concentration (>200 M), as runoff should be slower and trophic interaction tighten as residence time increases. Conclusions Results Sulfide concentration of the Hedionda is around 40M in winter and above 100M in Summer. Oscillatoria sp. is the predominant alga throughout the year, but highest concentrations are linked to sulfide concentration between 100-200M. This could be due to (i) advantages in growth rate at elevate sulfide concentration (poster 646), (ii) avoiding top down control by greater sized aggregate building and (iii) diminishing algal loss in a fluvial system by getting trapped on plants and branches. References 1. Beutler, M.; Wiltshire, K.H.; Meyer, B.; Moldaenke, C.; Lüring, M.; Meyerhöfer, U.-P.; Hansen, H. D. A fluorometric method for the differentiation of algal populations in vivo and in situ. Photosynth. Res. 2002, 72, pp. 39−53. doi:10.1023/A:1016026607048 2. Leboulanger, C.; Bouvy, M.; Carré, C.; Cecchi, P.; Amalric, L.; Bouchez, A.; Pagano, M.; Sarazin, G. Comparison of the effects of two herbicides and an insecticide on tropical freshwater plankton in microcosms. Arch. Environ. Contam. Toxicol. 2011, 61, pp. 599−613. doi: 10.1007/s00244-011-9653-3 3. Reul, A.; Muñoz, M.; Bautista, B.; Neale, P.J.; Sobrino, C.; Mercado, J.M.; Segovia, M.; Salles, S.; Kulk, G.; León, P.; van de Poll, W.H.; Pérez, E.; Buma, A.; Blanco, J.M. Effect of CO2, nutrients and light on coastal plankton. III. Trophic cascade, size structure and composition. Aquat. Biol. 2014, 22, pp. 59-76. doi: 10.3354/ab00585 4. Metorological Station of Estepona (Access 15/06/2017) http://www.juntadeandalucia.es/agriculturaypesca/ifapa/ria/servlet/FrontController?action=Static&url=coordenadas.jsp&c_provincia=29&c_estacion=4 Temporal evolution of mean air temperature an radiation (Fig. 2) shows an annual cycle where radiation proceed temperature. Temperatures above 20ºC were registered between June and November. Precipitation (Fig. 3) was absent in summer (June-September) and some small precipitation were observed in October-November, before considerable precipitation occurred in December. Sulfide concentration (Fig. 3) higher than 100 M were observed during the warm (>20ºC) and dry season, which dropped down to 40 M after the registration of the first precipitations. Low sulphide concentration maintained from December to May, and increased again in the last sampling date (30/05/2017). Highest chlorophyll a concentration (11 g Chl a /l) was observed at (27/06/2016, Fig. 4). In general Chl a concentration was significant higher (Median=1.8g/l) during the dry season when sulfide concentration was above 100 M, than when it was bellow 100M (Median=0.5g/l) (P<0.004 MannWhitney Rank Sum Test). Phtoplankton community was dominated by bluegreen algae (in our case Oscillatoria sp.) throughout the year, and specially during the dry, sulphide-rich season (80% of chlorophyll corresponded to bluegreen algae). Abundance and biovolume of Oscillatoria sp. (<100m) was highest at sulfide concentration between 100 and 200 M (Fig. 5). Considering the whole planktonic community (including aggregates of Oscillatoria sp. >100m), highest concentration of Oscillatoria sp. was observed at sulfide concentration between 100 and 200 M. Time from first sampling date abr ago dic abr Radiation (MJ/m2) 0 5 10 15 20 25 30 35 Radiation abr ago dic abr Temperature (ºC) 0 5 10 15 20 25 30 35 Temperature Fig. 2 Temporal evolution of mean air temperature and radiation during the sampling period. ( Time period of temperature >20ºC) Fig. 3 Temporal evolution of precipitation and sulfide concentration in the samples of hediond. ( dry, sulfide-rich season) Fig. 4 Temporal evolution of total Chl a concentration and relative contribution of different groups discriminated by fluorescence fingerprint. ( dry and sulfide rich season). Fig. 5 Biovolume and abundance of Oscillatoria sp. versus sulfide concentration. Fig. 6 Chlorophyll a concentration of bluegreen algae versus sulfide concentration. Acknowledgments •This work has been financially supported by the Spanish Ministry of Science and Innovation through CGL2014-53682-P project.