TheThe RoleRole ofof thethe Endosymbiotic,Endosymbiotic, NitrogenNitrogen Fixing,Fixing, CyanobacteriumCyanobacterium RicheliaRichelia intracellularisintracellularis inin thethe MediterraneanMediterranean SeaSea ParadoxParadox

Bar Zeev Edo, Yogev Tali, Aharonovich Dikla, Beja Oded and Berman-Frank Ilana The Paradox

The ultra oligotrophic ocean with the abnormal Redfield ratio ( N>>>P ) are explained by geochemical and geophysical aspects.

What is the biological explanation ???

A paradox is an apparently true statement or group of statements that leads to a contradiction or a situation which defies intuition The latest on N2 fixation in the

Papers by Nicole. Garcia et al (C. R. Biologies 329. 2006) and Andrew P. Rees et al (G. R. VOL. 33. 2006) suggest that fixation is a process that occurs in the Mediterranean Sea but :

With no description of the players and the amount of influence they have on the N- cycle of the Eastern Mediterranean Sea. The Eastern Levantine basin f pd . c

ir Eastern Levantine basin yc inson/PAPERS/enc ob r rd.edu/~ va r deas.ha . people

Israel

gif.b_planning_med/imgwww.cls.fr/mater/ Micro Nutrients in the eastern Mediterranean Sea

Ammonium, Nitrite Above DCM : Phosphate was barely detectable (3-6nM) DCM Ammonium was much higher (65-70nM)

At the base of the DCM (130-160m): Nitrite reached a sharp maximum (~70nM) * Silicate was present throughout the photic zone (~1.2µM)

Krom, M. D., E. M. S. Woodward, et al. (2005) Marine Nitrogen cycles

In oligotrophic regions primary In tropical Atlantic and Pacific production is often dominated by N - 2 ocean N2 fixation is responsible for fixing which favored 36- 50% of the total N demand for N-depleted surface waters. the microbial community. N fixation increases the N: P ratio 2 It has been estimated that N2 above Redfield ratio, which fixation is quantified over 50% of eventually produce nitrogen enriched the NO3 fluxes in those areas. waters.

Arrigo, K. R. (2005). "Marine microorganisms and global nutrient cycles (vol 437, pg 349, 2005)." Nature 438(7064): 122-122. Fast Induction and Relaxation (FIRe) system The FIRe system provides information on the photophysiological state of the phytoplankton and inflects on their condition. When Fv/Fm is high the phytoplankton’s condition is well, that means that the amounts of nutrients are sufficient and the rest of the physical and chemical elements are most suitable.

a. b. Our research objectives

1. To locate and quantify the cyanobacterium, Richelia intracellularis in the Eastern Mediterranean basin.

2. To identify the organisms by morphological and by their genetics characteristics and to compare them to related species around the world.

3. To follow the yearly cycle and changes in abundance of Richelia intracellularis at two stations off the of Israel (four depths in each station).

4. To assess their contribution to the N cycle and their role in the creation of the Mediterranean Sea's unusually high N: P ratios.

With / plus Life (Greek)

Definition : The interaction between two organisms when at least one of them benefits.

Parasitism (+ −) Commensalism (+ 0) Mutualism (+ +)

jpg.008imagewww.cbu.edu/.../ jpg.002imagewww.cbu.edu/.../ www.hawaiisfishes.com Symbiosis

The relationship can be divided into: Endosymbiosis - which the symbiont lives inside its . Ectosymbiosis - which the symbiont lives outside its host ( on the surface of the host or inside its digestive tract ).

endosymbiosis ectosymbiosis

30µm 40µm

Edo Bar-Zeev Shigenobu ,Takeda Ken; ,Furuya ;Gómez, Fernando asp.thumbnailswww.elgal.co.il/site/ The relationship between the host and its companion

carbohydrates cyanobiont

• Buoyancy • Protection from grazers • Fixed C as carbohydrates • Stable environment and a wider nutrient pool

ATP

N2 fixation

Fixed N as ammonia nitrite or nitrate Early descriptions of phytoplankton in the Eastern Mediterranean Sea

• From the early 1970's Prof. B. Kimor has described the existence of several symbiotic associations such as Rhizosolenia-Richelia and Hemiaulus-Richelia.

• His identification of these species in the Eastern Mediterranean Sea and the current focus on their importance as contributors of new N in N-depleted regions ( Rachel A Foster and Jonathan P. Zeher - E. M. 8. (2006) and Edward J. Carpenter et al - M.E.P.S. 185. (1999), lead to our current study. Who is the Mediterranean Richelia intracellularis b a

30µm c d Heterocyst

40µm

The right panel shows the diatom Rhizosolenia spp and its endosymbiotic cyanobacterium- Richelia intracellularis. The left panel shows the Hemiaulus hauckii diatom and its endosymbiotic cyanobacterium- Richelia intracellularis. f- combined picture of all the sets of filters and bright field microscopy. a.g- organisms under bright field light. b.h- organisms under chlorophyll fluorescence (ex: 470 nm, em: 680 nm). c.i- organisms under phycocyanin fluorescence (ex: 620, em: 660 nm). d.j- organisms under phycocyanin fluorescence (ex: 490 nm, em: 580 nm ). Initiation and maintaining the symbiosis

The symbiosis in lasts several generations. They usually acquire their cyanobionts from the parent cells during division. even though they are not fully synchronized.

a. b. c. by diatom/cyanobacterial symbiosis in the tropical • Carpenter, E.J et al are showing that a significant amounts of “new nitrogen” (0.45Tg 10d-1 ) enter the N cycle duo to nitrogen fixation by Richelia intracellularis. • They also show the correlation between the number of Richelia cells to the

N2 fixation

Cruise track by the RV ‘Seward Jonson’ November 1996

Carpenter, E. J., J. P. Montoya, et al. (1999) The correlation between Nitrogen fixation and the No of Richelia cells

deep station: N fixationRichelia and cells shallow station: N fixationRichelia and cells -1 300 -1 -1 400 -1 80 100 d 350 d 250 70 90 -2 300 -2 80 60 200 cells L cells 70

250 L cells 50 200 150 60 40 50 150 c 30 100 40 100 30 20 50 20 umol N m umol 50 10 Nm umol Richelia 10 Richelia 0 0 0 0 A pr-06 Jun-06 A pr-06 Jun-06 Oct-06 Nov-06 Oct-06 Nov-06 Feb-06 M ar-06 Feb-06 M ar-06 Sep-06 Sep-06 May-06 May-06 month month N fixation Richelia N fixation Richelia cells

It seems like there is a good correlation between the abandons of richelia cells and the Nitrogen fixation according the Acetylene Redaction technique Molecular analysis of the Mediterranean Richelia intracellularis Phylogenetic reconstruction of nifH sequences obtained from the Levantine basin. The rest of the Richelia intracellularis The scale bar represents 0.1 nifH population substitutions per site and one sequence of erythraeum (IMS101) was used as the 5% 54.5% 45. outgroup.

The sequences ratio between populations with nifH obtained in the Eastern Mediterranean Sea Richelia Conclusions intracellularis and Work plan- 2007

1. We know they are out there 2. We know they have the potential of injecting large amounts of “New Nitrogen“ in to the aquatic system 3. We know they do fix nitrogen in the Levantine basin

How much, when exactly and where…. Until now, 2004 - 2006 Nitrogen fixation in the Eastern Mediterranean Sea

BiologicalCluster NI fixation is clearly present in the ClusterEastern II Mediterranean Sea. Cluster III its impact on the N cycle is unclear.

N-fixation rates Solvingvolumetric range this- 0.4 is- 3.05 our nmol N goalL-1d-1 Integrated values - 130-357 µmol N m-2 d-1 Acknowledgments

We would like to thank : • The Captain (Sheffer Dani) and Engineer (Catzman Etai) of the R/V Mediterranean Explorer . • EcoOcean Marine Research and Education Organization • Captain and crew of the R/V Shikmona • Rahav Eyal, Cimand Oran , Robin Maxim, Vafrik Anna and Rosen Yoav for technical help during and after the cruises •NuritKress •HerutBarak • ISF funding to I. B-F. and O. B.

Trichodesmium ??? Deep station Deep station Deep station Deep station Deep station Deep station -N fixation rates

Integrated values - 130-357 µmol N m-2 d-1

volumetric range - 0.4 - 3.05 nmol N L-1d N-fixation rates volumetric range - 0.4 - 3.05 nmol N L-1d-1 Integrated values - 130-357 µmol N m-2 d-1 Nutrient exchanges (N and C) and the distribution pattern of glutamine synthetase (GS)