Py idine nucleo ide le els in
zooplank on: labo a o y
and ield samples
Ciclo de Ciencia Compa ida, 12 Junio 2012
Osma N., Packa d T. and Gómez M.
Ins i u e o Oceanog aphy and Global Change, Biological Oceanog aphy G oup, Uni e si y o Las Palmas de G an Cana ia, Cana y Islands, Spain.
E-mail add ess: nosma@beca ios.ulpgc.es
Respi a ion
- Ubiqui ous p ocess
- Key componen in he es ima ion
o he ca bon lux.
Ne e heless…
¿¿OCEANIC RESPIRATION ??
Del Gio gio and Dua e, 2002
Due o
- Di icul o quan i y in si u espi a ion a es.
- Di ec measu emen s  Incuba ions
SOLUTION!! Biochemical assays
Po en ial Respi a ion (F)
Elec on anspo sys em (ETS) ac i i y o es ima e espi a o y oxygen consump ion (RO2)
Packa d e al., 1971
Vmax
Subs a e sa u a ion
Wha mechanism con ols he RO2?
Physiological RO2 ETS ac i i y
Packa d e al., 1996
Ho: In i o po en ial espi a ion de e mined by he Vmax and he subs a es
a ailabili y .
RESPIRATION MODEL
BASED ON SUBSTRATE LIMITATION
Py idine nucleo ides and succina e
Py idine Nucleo ides (PNs)
NAD NADP
(Be ge e al., 2004; Ziegle , 2005; Pollack e al., 2007; Ying, 2008)
NAD+ NADH
Ca abolism
NADPH
NADP+
Anabolism
 Reducing equi alen s o hund ed o cellula eac ions.
 Func ions:
- Modula ing ene gy me abolism, educ i e biosyn hesis and an ioxida ion p ocesses.
- Recen s udies, impo ance in cell signaling in animals, plan s and ungi.

P e ious Wo k
 Few s udies in ma ine o ganism
Animal physiology
- Du ing 50’s and 60’s, de elopmen o di e en echniques o measu e PNs (Chance e al.,1955,1962;
Low y e al. 1961).
- Recen clinical applica ions (Maye sky and Chance, 2007).
Plan physiology
- Knowledge inc easing s eadily  Le els and oles unde di e en me abolic condi ions.
(Molle and Rasmusson, 1994; Hage don, 2004; Noc o e al., 2006)
Bac e ial ac i i y
- Wos and Polla d (2009): NADH as an index o bac e ial me abolic ac i i y in ac i a ed sludge.
- NADP+ in ela ion o isoci a e dehyd ogenase ac i i y (Roy and Packa d, 1998).
- A emp ed o es ima e ma ine p ima y p oduc ion wi h he NADPH (S eigenbe ge e al., 2004), .
P e ious Wo k
 Few s udies in ma ine o ganism
Animal physiology
- Du ing 50’s and 60’s, de elopmen o di e en echniques o measu e PNs (Chance e al.,1955,1962;
Low y e al. 1961).
- Recen clinical applica ions (Maye sky and Chance, 2007).
Plan physiology
- Knowledge inc easing s eadily  Le els and oles unde di e en me abolic condi ions.
(Molle and Rasmusson, 1994; Hage don, 2004; Noc o e al., 2006)
Bac e ial ac i i y
- Wos and Polla d (2009): NADH as an index o bac e ial me abolic ac i i y in ac i a ed sludge.
- NADP+ in ela ion o isoci a e dehyd ogenase ac i i y (Roy and Packa d, 1998).
- A emp ed o es ima e ma ine p ima y p oduc ion wi h he NADPH (S eigenbe ge e al., 2004), .
Up o now, NO wo k in ma ine o ganisms
Desc ibing in acellula le els o PNs
Compa ing PNs le els wi h espi a ion a es
Labo a o y Wo k
Oxy his ma ina
He e o ophic dino lagella e.
High ole ance o change in empe a u e,
salini y and nu ien s  Good o cul u ing!
Why??
 Easy o cul u e
 Model o ganism  espi a o y me abolism??
D oop,1959; Kimmance e al., 2006; Jeong e al., 2003
Oxy his ma ina,
A is izabal (2009)
20
µm Rhodomonas salina
 Fed wi h Rhodomonas salina
 Same cul u ing condi ions, excep o ligh
Field Samples

C uise ack o he R/V Hespe ides du ing he LEG 3, 4 and 7.
•LEG 3,4: Feb ua y - Ap il 2011.
•LEG 7: June - July 2011.
Sampling
Me hods
SIZE FRACTIONATION
150 - 200 m
sampling
100 – 500 µm
500 – 1000 µm
> 1000 µm
100 µm mesh size WP-2 ne
0 h: Well ed
24 h: S a ed
RESPIRATION MEASUREMENTS
ON BOARD
S o age a - 80oC
LABORATORY WORK AT INSTITUTE
ETS ACTIVITY
INTRACELLULAR SUBSTRATES
PROTEIN MASS
Resul s & Discussion
Speci ic espi a o y oxygen consump ion dec eases wi h size ac ion, bo h in well- ed and
s a a ion condi ions.
No signi ican ly di e ences o he R/F a io be ween well- ed and s a ed o ganisms.
Resul s & Discussion
100-500 µm 500-1000 µm
> 1000 µm Speci ic NADH and NADPH concen a ion no
signi ican ly di e en in he h ee size classes.
No signi ican ly di e ences be ween well- ed
and s a ed o ganisms,
- Excep ion: NADH le els in 100-500 m size ac ion
Conclussions

Conclussions
The all in he espi a ion du ing he onse o s a a ion sugges s ha espi a ion
is subs a e limi ed du ing his pe iod.
To al PNs and espi a ion a e well co ela ed du ing s a a ion in a ma ine
dino lagella e. This obse a ions suppo s he use o a espi a ion model based
on subs a e limi a ion.
Labo a o y wo k …
ENZYME KINETIC MODEL
Ro =
Vmax: Maximum eloci y o he eac ion
S1, S2: Subs a e concen a ion o NADH and NADPH
K1, K2: Michaelis cons an o NADH and NADPH
Kia: Appa en dissocia ion cons an
𝑉𝑚𝑎𝑥 · [𝑆1 ·𝑆2]
𝐾1 · 𝐾𝑖𝑎 + 𝐾2 · [𝑆1] + 𝐾1 · [𝑆2] + [𝑆1 ·𝑆2]
Packa d and Gomez, 2008
Field da a …
E ec o s a a ion seen in 100-500 µm size class. Highes size classes migh
ha e a la ge ese e and 24 h o s a a ion migh no challenge hem.
Acknowledgemen s
This esea ch is amed in he EXZOME p ojec (CTM 2008 – 01616/MAR), which is
unded by he ex inc Spanish Science and Educa ion Minis y. N. Osma ecei es
inancial suppo om he Fo ma ion and Pe ec ion o he Resea che Pe sonal
P og am om he Basque Go e nmen .
I also hank o MALASPINA 2010 (CSD-20080077) p ojec o in i ing me o
pa icipa e in i s c uise.
Py idine nucleo ide le els in
zooplank on: labo a o y
and ield samples
Ciclo de Ciencia Compa ida, 12 Junio 2012
Osma N., Packa d T. and Gómez M.
Ins i u e o Oceanog aphy and Global Change, Biological Oceanog aphy G oup, Uni e si y o Las Palmas de G an Cana ia, Cana y Islands, Spain.
E-mail add ess: nosma@beca ios.ulpgc.es