Pyridine nucleotide levels in zooplankton: laboratory work and field samples

Pyridine nucleotide levels in zooplankton: laboratory and field samples Ciclo de Ciencia Compartida, 12 Junio 2012 Osma N., Packard T. and Gómez M. Institute of Oceanography and Global Change, Biological Oceanography Group, University of Las Palmas de Gran Canaria, Canary Islands, Spain. E-mail address: [email protected] Respiration - Ubiquitous process - Key component in the estimation of the carbon flux. Nevertheless… ¿¿OCEANIC RESPIRATION ?? Del Giorgio and Duarte, 2002 Due to - Difficult to quantify in situ respiration rates. - Direct measurements  Incubations SOLUTION!! Biochemical assays Potential Respiration (F) Electron transport system (ETS) activity to estimate respiratory oxygen consumption (RO2) Packard et al., 1971 Vmax Substrate saturation What mechanism controls the RO2? Physiological RO2 ETS activity Packard et al., 1996 Ho: In vivo potential respiration determined by the Vmax and the substrates availability . RESPIRATION MODEL BASED ON SUBSTRATE LIMITATION Pyridine nucleotides and succinate Pyridine Nucleotides (PNs) NADt NADPt (Berger et al., 2004; Ziegler, 2005; Pollack et al., 2007; Ying, 2008) NAD+ NADH Catabolism NADPH NADP+ Anabolism  Reducing equivalents for hundred of cellular reactions.  Functions: - Modulating energy metabolism, reductive biosynthesis and antioxidation processes. - Recent studies, importance in cell signaling in animals, plants and fungi. Previous Work  Few studies in marine organism Animal physiology - During 50’s and 60’s, development of different techniques to measure PNs (Chance et al.,1955,1962; Lowry et al. 1961). - Recent clinical applications (Mayevsky and Chance, 2007). Plant physiology - Knowledge increasing steadily  Levels and roles under different metabolic conditions. (Moller and Rasmusson, 1994; Hagerdon, 2004; Noctor et al., 2006) Bacterial activity - Wos and Pollard (2009): NADH as an index of bacterial metabolic activity in activated sludge. - NADP+ in relation to isocitrate dehydrogenase activity (Roy and Packard, 1998). - Attempted to estimate marine primary production with the NADPH (Steigenberger et al., 2004), . Previous Work  Few studies in marine organism Animal physiology - During 50’s and 60’s, development of different techniques to measure PNs (Chance et al.,1955,1962; Lowry et al. 1961). - Recent clinical applications (Mayevsky and Chance, 2007). Plant physiology - Knowledge increasing steadily  Levels and roles under different metabolic conditions. (Moller and Rasmusson, 1994; Hagerdon, 2004; Noctor et al., 2006) Bacterial activity - Wos and Pollard (2009): NADH as an index of bacterial metabolic activity in activated sludge. - NADP+ in relation to isocitrate dehydrogenase activity (Roy and Packard, 1998). - Attempted to estimate marine primary production with the NADPH (Steigenberger et al., 2004), . Up to now, NO work in marine organisms Describing intracellular levels of PNs Comparing PNs levels with respiration rates Laboratory Work Oxyrrhis marina Heterotrophic dinoflagellate. High tolerance to change in temperature, salinity and nutrients  Good for culturing! Why??  Easy to culture  Model organism  respiratory metabolism?? Droop,1959; Kimmance et al., 2006; Jeong et al., 2003 Oxyrrhis marina, Aristizabal (2009) 20 µm Rhodomonas salina  Fed with Rhodomonas salina  Same culturing conditions, except for light Field Samples Cruise track of the R/V Hesperides during the LEG 3, 4 and 7. •LEG 3,4: February - April 2011. •LEG 7: June - July 2011. Sampling Methods SIZE FRACTIONATION 150 - 200 m sampling 100 – 500 µm 500 – 1000 µm > 1000 µm 100 µm mesh size WP-2 net 0 h: Well fed 24 h: Starved RESPIRATION MEASUREMENTS ON BOARD Storage at - 80oC LABORATORY WORK AT INSTITUTE ETS ACTIVITY INTRACELLULAR SUBSTRATES PROTEIN MASS Results & Discussion Specific respiratory oxygen consumption decreases with size fraction, both in well-fed and starvation conditions. No significantly differences of the R/F ratio between well-fed and starved organisms. Results & Discussion 100-500 µm 500-1000 µm > 1000 µm Specific NADH and NADPH concentration not significantly different in the three size classes. No significantly diferences between well-fed and starved organisms, - Exception: NADH levels in 100-500 m size fraction Conclussions Conclussions The fall in the respiration during the onset of starvation suggests that respiration is substrate limited during this period. Total PNs and respiration are well correlated during starvation in a marine dinoflagellate. This observations supports the use of a respiration model based on substrate limitation. Laboratory work … ENZYME KINETIC MODEL Ro = Vmax: Maximum velocity of the reaction S1, S2: Substrate concentration of NADH and NADPH K1, K2: Michaelis constant of NADH and NADPH Kia: Apparent dissociation constant 𝑉𝑚𝑎𝑥 · [𝑆1 ·𝑆2] 𝐾1 · 𝐾𝑖𝑎 + 𝐾2 · [𝑆1] + 𝐾1 · [𝑆2] + [𝑆1 ·𝑆2] Packard and Gomez, 2008 Field data … Effect of starvation seen in 100-500 µm size class. Highest size classes might have a larger reserve and 24 h of starvation might not challenge them. Acknowledgements This research is framed in the EXZOME project (CTM 2008 – 01616/MAR), which is funded by the extinct Spanish Science and Education Ministry. N. Osma receives financial support from the Formation and Perfection of the Researcher Personal Program from the Basque Government. I also thank to MALASPINA 2010 (CSD-20080077) project for inviting me to participate in its cruise. Pyridine nucleotide levels in zooplankton: laboratory and field samples Ciclo de Ciencia Compartida, 12 Junio 2012 Osma N., Packard T. and Gómez M. Institute of Oceanography and Global Change, Biological Oceanography Group, University of Las Palmas de Gran Canaria, Canary Islands, Spain. E-mail address: [email protected]