Christophe Brunet Stazione Zoologica A. Dohrn Napoli -

Transcription

1 MARBEF Advanced course The role of flow cytometry in Marine biodiversity and ecosystem functioning Ecophysiological approaches for understanding ecosystem functioning: integrating flow cytometry and environmental data Christophe Brunet Stazione Zoologica A. Dohrn Napoli - 6 /11/04;

2 The content of this presentation is the exclusive property of its author. Any use is prohibited. If you wish to use any material for any purpose whatsoever, permission must be obtained from the author.

3 THE ROLE OF FLOW CYTOMETRY IN MARINE BIODIVERSITY AND ECOSYSTEM FUNCTION UNTREATED WATER FLUORESCENT DYE PRESERVED WATER INPUTS FLUORESCENT PROBE OUTPUTS2 SORTING UPTAKE X X X X FLOW CYTOMETER X DATA CONVENTIONAL ANALYSIS OUTPUTS1 MULTI- VARIATE ANALYSIS NOVEL ANALYSIS (Images/pulses) Species A thingy B wotsit C blob.... Z bug Abundance TIME GENERAL TARGETTED COMMUNITY BIODIVERSITY COMMUNITY/ POPULATION ACTIVITY FLUOR. FLUOR. RATE X X X X X LIGHT SCATTER COMMUNITY ABUNDANCE LIGHT SCATTER POPULATION ABUNDANCE CONCENTRATION COMMUNITY/ POPULATION PROCESSES

4 The role of flow cytometry in Marine biodiversity and ecosystem functioning Overview of the presentation Photoacclimation : What is it? Studying photoacclimation of algae: parameters, scales and examples Relationships between photoacclimation and physical characteristics

5 Phytoplankton - Biomass, Biodiversity, Growth rate, Productivity (= photosynthesis) - Need of Light, Nutrients, Carbon production (and flow) depends on the interactions B L - N depend on, for a great part, the physical environment mixing, stratification, waves,

6 Light variations in the environment : Different scales of variations : - Diel cycle - Cloud cover (regional scale, wind forcing, ) - Seasonal change : photoperiod, intensity Marine environment peculiarities: - Water mass characteristics (more or less rich in SM, coast,...), - Tidal cycles (14 days, 12 h), - Turbulence, - Migrations, - Advections of biomass

7 P E Light is limitant Light is optimal Light is high Light is photoinhibiting Continuously need of acclimation (in time and space)

8 Photo-acclimation : Responses to light variations To optimize photosynthesis under different light conditions experienced by cells Ecological : active displacements of cells,... BioPhysical : fluorescence (energy dissipation) Physiological : cell cycle,... Biochemical : pigment changes, proteins, lipids (permeability of membranes, ), enzymatic reactions Cell Biology : morphological changes Molecular : (de-) activation of genes (pigments, proteins, lipids...)

9 Photoacclimation : To low light : Increase of light-harvesting capabilities (e.g. quantity of pigments) To high light : Reducing the light-harvesting capability, and increasing the dissipating rate of energy (NPQ) Photoprotection: to protect chls. and other components of the PSII. Photoinhibition: light-dependent reduction of photosynthetic efficiency Photostress: non-adaptative strategy.

10 How to dissipate excess of energy? (PSII) Light Fluorescence E heat Membrane of thylakoid LHCs P 680 Pigment: Xanthophyll cycle Non-photochemical quenching k P Photochemistry From Vantrepotte and Brunet, Phyto-Pam report

11 Studying photoacclimation of algae: parameters, scales and examples Parameters : # Variable fluorescence (PAM, Pump&Probe) # Passive fluorescence / chla # other pigments

12 Fm For dark adapted samples Yield = Fv/Fm Fv Maximal fluorescence Fo luo. signal Saturating (pump) Close the RCs Mes. Freq. Light flashes Probe flash Probe flash Variable fluorescence (PSII) Example for Phyto-Pam functioning (Walz) From Vantrepotte&Brunet

13 Coupling between Flow cytometry & Pump & Probe fluorescence Some refs: Olson & Zettler, 1995, L&O 40 Olson, R.J., et al Cytometry, 37 Olson, R.J.et al Deep-Sea Research II 47: Shalapyonok, A. et al Deep-Sea Research II 48: Measurements of Fv/Fm on single cells (# whole community) Inconvenient : low fluorescence signals for many small species need of population averaging of signals

14 ttp:// Methods: DP flow cytometer : time course of Chl fluorescence yield during a 50 µs excitation flash. Fm Fo DP microfluorometer, individual cells in a sample chamber are isually identified, and fluorescence excitation is provided by a lue light-emitting diode which can be configured to provide a aturating flash and also a subsequent series of short flashlets. his sequence allows both saturation and relaxation kinetics to be monitored. P.D.P: not Paperone de Paperoni (PICSOU / SCROOGE) but Pump during Probe

15 Schema of the PDP microscope ttp://

16 Optical layout of the PDP flow cytometer

17 Olson et al., website PDP flow cytometric analysis of the phytoplankton in a water sample from 30 m depth south of the Polar Front in the northern Ross Sea.

18 Pump-during-probe microfluorometry of Fragilariopsis spp. Olson et al., website Reference signal from flash sequence. Cell fluorescence Fluorescence yield

19 Effect of iron enrichment on different groups

20 Very powerful tool, allowing to better describe the PP variability at sea as well as to understand succession / competition processes Fv/Fm ratio for different groups (in the same environmental conditions) Note the difference between the whole comunity (FRR) and separated groups H.M. Sosik, R.J. Olson / Deep-Sea Research I 49 (2002)

21 Photoacclimation Passive fluorescence Kiefer D.A., Mar.Biol.22 Kiefer D.A., Mar Biol. 23 Kiefer DA & Reynolds RA Red fluorescence of FC : chla fluorescence by cells chla quantity and its photoresponse Photoresponse: decrease of fluorescence under high light and vice-versa

22 % variations HL to LL Phaeodactylum LL to HL Red Fluorescence Culture exp. Red increases from HL to LL and vice-versa LL to HL % variations Time after light shift HL to LL Rhodomonas Changes in chla C? or physiological acclim.? Casotti, Brunet, Dimier, in prep

23 Conclusions: Red fluo. as indicator of photoacclimation In some species, it is related to photoprotective features (at short term scale) (however, be careful to photoinhibition processes (Fo, Fm))

24 In situ Photoacclimative signal along the water column for Prokaryotes (Syn, Prochl.): Strong increase in Red and Orange fluo with depth (See other talks and many publications) Gulf of Naples, Casotti et al., 2000

25 Coupling between F. Cytometry data and Pigments (HPLC) In situ: Veldhuis & Kraay, 1990 Veldhuis et al., 1993 Veldhuis & Kraay, 2004 Brunet et al., 2003 Casotti et al., Cultures: Partensky et al., 1993 Morel et al., 1993 Moore et al., 1995 Moore & Chilsom, Target species: Prochlorococcus -Dvchla pigment - Strong photoacclimation responses to light variations (along the water column) - Physiologically distinct ecotypes (Moore et al., 1998; Bibby et al., 2003) along the water column (with different red fluo values)

26 Examples: - Strait of Sicily, July 1997 Prochlorophytes : Relationships between cell number and Dvchla Station 11 (5 m depth, filament) 0.1 > 50 meters Dvcha = 1.415e-06 * cells ; r 2 = 0.92 good tracers of water masses (Dusenberry et al 2000) Dvcha fg ml < 50 meters dvcha = 2.74E-07 * cells r 2 =0.90 Mean content of dvchla 0.44 fgcell -1 (SD: 0.62) 1.56 fgcell -1 (SD: 0.54) Prochloro cells ml -1 Brunet/Casotti et al., in prep

27 Temperature (5 m depth, C) Sicily ST46 ST53 Station ST13 ST11 ST7 Lat. N ST15 ST18 ST Front / Filament sytem Long. E

28 In situ Diel cycle : no clear signal of photoacclimation for the three populations discriminated by FC! strong signal of division (daily pattern) with increase of pigment content during the day before the division (Dusenberry et al., 2001; Jacquet et al., 2001; Claustre et al, 2002)

29 Red fluo. Prochlorophytes depth (m) To remove the diel periodicity due to cell growth Time (hours) Gamma parameter (Dusenberry et al., 1999) Red/(FALS) is the exponent of the power law relationship Gamma increases with depth and decreases during the day depth (m) Gulf of Naples Brunet/Casotti in prep Time (hours)

30 elationships between photoacclimation and physical characteristics Relationships between photodependent parameters and light Compare between different ecosystem (// light, mixing,...)

31 elationships between photoacclimation and physical characteristics Photodependent parameters as light history markers of cells Aims: Lewis et al 1984 Cullen & Lewis, 1988 Dusenberry&Chisholm1999 Dusenberry, 2000 Dusenberry et al Dusenberry et al 2001 Brunet et al Casotti et al tracers of vertical mixing in the upper layer - physiological responses to mixing, Productivity If mixing is low, photoacclimation takes place and vice-versa...

32 Light (W/m 2 ) Fv/Fm ratio Depth (m) r 2 =0.996 Irrad = 595xexp(0.22xZ) r 2 = Fv/Fm = -0.28xexp(0.18xZ)+0.52 Gulf of Naples Mean of daily data Brunet etal in prep Dd/Chl.a ratio Dt/Chl.a ratio Dt/(Dt+Dd) ratio Depth (m) r 2 =0.998 Dd/Chl.a = xexp(0.0883xZ) r 2 =0.983 Dt/Chl.a = xexp(0.0849xZ) r 2 =0.987 Dt/(Dt+Dd) = 0.184xexp(0.0978xZ) but it depends on the reactivity of the parameter, so on the temporal scale

33 Example from the Gulf of Naples Ln (Fv/Fm) Ln (Irradiance) r²= 0.82, n = 60 7 Latitude Nord Posillipo 6 9 Cycle N A P O L I Longitude Est F.Sarno Irradiance (W.m -2 ) Fv/Fm ratio Ln (Dt/(Dt+Dd) r²=0.70, n =30 Ln (Dt/(Dt+Dd) r²=0.57, n= Brunet et al. 2003

34 From incubation experiments (light from 10% to 90% of Io), + In situ data in the upper mixed layer (diff. between 2 and 20 m) stimate the vertical velocity of cells in the mixed layer (2 to 20 meters depth) Velocity was about 0.07 cm.s -1

35 So, we can determine the % of change of other parameters in th same mixing layer Velocities of changes are different in function of the considered parameters

36 From these estimations: Parameters varying at < 4%h -1 : no signif. difference between 2 and 20 m So 4%h -1 is the lower threshold for photoacclimation rate So for this value : mixing rate = photoacclimation rate (Lewis et al., 1984) Kv / l 2 r = Kl l: mixed layer, (K attenuation coeff., l: mixed layer) r: photoaccl. rate From this equation, we can estimate the vertical eddy diffusivity (Kv) Kv = m 2 s- 1

37 Mixed layer dynamics Photoacclimation relationships - Dusenberry et al (1999,2000,2001) Two models can be applied: * First order kinetic * Logistic (Cullen & Lewis 1988) Logistic: Xt = K* Xt * ((1 Xt)/Xen)) Logistic model allows to parameterize hysteresis in acclimation rates (which represents the history dependence of the response, with changes effect when forces are changed) leading to faster acclimation for a shift to HL that the reciprocal shift (Dusenberry, 2000) Aim and advantage of these studies : * to simulate photoacclimation, photosynthesis, specific growth rate in the upper layer in function of depth (and time) by respect to different mixing features (so different vertical diffusivity) * Single-cell properties Constraints: diel cycle, photodependent parameters varying at time scale of few hours or less

38 Some results (Dusenberry, 2000): - Variance of photoacclimative property maximal at intermediate mixing rates and small at low and high mixing rates; - FOKM and LM : photosynthesis maximal at low vertical diffusion rates - FOKM: Growth rate maximal at low rates of diffusion - LM: Growth rate maximal at high rates of diffusion difference due to hysteresis, which could play an important role on photoacclimation rate