Viruses :Conductors of aquatic ecosystems ?

Stéphan JACQUETThonon-les-Bains

Outline

The Group of Aquatic Microbial Ecology

The Importance of Aquatic Ecosystems

The Importance of Aquatic Micro-organisms

The Importance of Aquatic Viruses

The Key Roles played by Aquatic Viruses

The Phage Therapy in Aquatic Environments

Take Home Messages

The Uncharacterized Viral Diversity

GAME

Group of Aquatic Microbial Ecology

Evaluate and study the diversity, the dynamics and the functioning of aquatic microbial communities,

from viruses to protozoan

The French Aquatic Viral Network

AQUAPHAGE

Relationships between prokaryotic and viral diversity in different aquatic environments

Genomics & Ecology of Aquatic Viruses

Banyuls-sur-Mer, France 11-13 February, 2008

A meeting in the context of the Marine Genomics Europe Network and RAVAGE

Réseau frAnçais de Virologie Aquatique de la Génomique à l'Ecologie

Aquatic Ecosystems

Aquatic habitats represent >70% of the Earth’ surface

>50% of the ocean is >3,000 m depth (V=1.3 x 109 km3)

Freshwater ecosystems represent 0.02% of the total water volume

The oceans control the climate, produce half of the Earth’s oxygen

Aquatic MicroorganismsMicroorganisms constitute >90% of living biomass in the sea

Total number of prokaryotes in aquatic habitats : 1.2 x 1029 cells~ similar to soils. Freshwater : 2.3 x 1026 cells

Total biomass of prokaryotes in aquatic habitats and oceanic sub-surfaces : ~ 60-100% of the total C found in plants

The higher cellular production of prokaryotes is found in aquaticecosystems : >1030 cells/year

Photosynthetic and heterotrophic microorganisms play a key rolein ecosystem functioning and the global biogeochemical cycles.Phytopk fix up to 50 GtC/year vs. BP averages 50% of the PP

Prokaryotes dominate over unicellular eukaryotes by a factor of2-3 orders of magnitude in the pelagic environment

In 1 ml of water samples (oceans, lakes, estuaries, etc) :

Heterotrophic prokaryotes 1 000 000 cellsPhotosynthetic prokaryotes 100 000 cellsProtozoan (Flagellates, Ciliates) < 10 000 cellsMicroalgae < 5 000 cellsZooplankton << 1Fishes 0 !!!

Aquatic Microorganisms

Quid of Viruses ?

Abundance

“ In the field of observation,chance favors the prepared mind ”

Louis Pasteur

Aquatic VirusesAbundance

Aquatic Viruses

Aquatic Viruses

« L´essentiel est invisible pour les yeux »

Antoine de Saint-Exupéry

Abundance

Aquatic Viruses

“ Everything is everywhere… ”

Lourens GM Baas Becking

Abundance

Spencer (1955) : Viruses in the sea but largely ignored for the next 35 years because of low abundances

Aquatic Viruses

Torella & Morita (1979) : Persuasive evidence of high viral abundances in the sea using TEM

Abundance

Torrella, F., and R. Y. Morita. 1979. Evidence by electron micrographs for a high incidence of bacteriophage particles in the waters of Yaquina Bay, Oregon: ecological and taxonomical implications. Appl. Environ. Microbiol. 37:774-778

… A minimum of 104 phage particles per ml was estimated…

… It is assumed that the actual number of phage particles is higher than 104 particles per ml…

…The implications of the presence of such concentrations in bays and estuaries with a certain level of eutrophication are of obvious importance in considering the microbial ecology of these environments…

Aquatic VirusesAbundance

Aquatic Viruses

Spencer (1955) : Viruses in the sea but largely ignored for thenext 30 years because of low abundances

Torella & Morita (1979) : Persuasive evidence of high viral abundances in the sea using TEM

Spencer (1955) : Viruses in the sea but largely ignored for the next 30 years because of low abundances

Bergh et al. (1989) : Demonstration of high viral concentrations in aquatic ecosystems,still using TEM

Abundance

Aquatic Viruses

Bergh, O., K. Y. BØrsheim, G. Bratbak and M. Heldal. 1989. High abundance of viruses found in aquatic environmentsNature 340: 467- 468

... Using a new method for quantitative enumeration, we have found up to 2.5 x I08 virus particles per millilitre in natural waters.

These concentrations indicate that virus infection may be an important factor in the ecological control of planktonic micro-organisms, and that viruses might mediate genetic exchange among bacteria in naturalaquatic environments.

Abundance

Bergh et al. (1989) : Demonstration of high viral concentrationin aquatic ecosystems,still using TEM

Aquatic Viruses

Spencer (1955) : Viruses in the sea but largely ignored for thenext 30 years because of low abundances

Torella & Morita (1979) : Persuasive evidence of high viral abundances in the sea using TEM

Spencer (1955) : Viruses in the sea but largely ignored for the next 30 years because of low abundances

Techniques improvements and fluorescent dyes : TEM EFM FCM MPNA, PA Q-PCR

Abundance

2x1011 stars in the milky way

1x1010 viruses per liter of water

>1030 viruses in aquatic habitats

Aquatic Viruses

Phages are probably the most abundant life forms on Earth

Abundance

The viral string of pearls is ~10 million light years long

Aquatic VirusesAbundance

From Suttle 2007

Viruses represent 5% of the prokaryotic biomass

Viruses contain more carbon than 75 million blue whales (~280 Mt)

Aquatic Viruses

The main domains of phage research

The environment (ecology and pollution)

The bacterial pathogenicity

The food industry

The evolution

The genomic aspect

The phagotheraphy

Lytic cycle

Lysogenic cycle

Nutriments, Temperature, host physiology

Insertion

UV, nutriments, mutagens,environmental stress

Diet

Diversity, population control, nutrient fluxes Character acquisition

Induction

Aquatic VirusesVirus life cycle

Aquatic VirusesVirus life cycle

The most important life cycle is still not known in aquatic habitats Contradictory results dealing with lytic vs. lysogenic processes: - important spatial and time variability - important shifts from one to another (environmental factors)

Viral-induced cell lysis has been the most studied to date

25-80% of viruses in a community are likely to be infectious

There are ~1023 viral infections per second in the ocean

20 to 60% of member species are lysogens (i.e. contain prophages)

The frequency of lysogeny varies among taxonomic groups

Aquatic Viruses

Assessing the role of livings in the functioning of aquatic ecosystems

require to be able to give answers to 3 basic questions :

Which organisms are there and in which proportion ?

What are organism metabolic and reproduction rates ?

What kind of players are they in the functioning of ecosystems ?

Aquatic Viruses

Geneva Lake

Lake Bourget

Prof

onde

ur (m

)

2004 2005 2006

Abundance, distribution, dynamics

High viral numbers are found in surface waters, in near-shore waters, in eutrophic waters, during productive seasons

10

20

30

40

50

1e+6 2e+6 3e+6 4e+6 5e+6 6e+6 7e+6

Mar May Jul Sep Nov

10

20

30

40

50

2.0e+7 4.0e+7 6.0e+7 8.0e+7 1.0e+8 1.2e+8

Heterotrophic Bacteria

Viruses

Cell ml-1

Part ml-1

Dep

th (m

)D

epth

(m)

r= 0.48

p= 0.03

n= 21

Aquatic Viruses

From Personnic 2007

Microbial loop

Classical chain

Zooplankton

Phytoplankton

Inorganic Nutrients Organic Nutrients

virus

Bacteria

Protozoans

Fishes

Aquatic Viruses

Ecological Role of Aquatic Viruses

E. h

uxle

yi (c

ell.m

l-1)

0

20x103

40x103

60x103

80x103

100x103

120x103

0

10x106

20x106

30x106

40x106

50x106

Viru

s de

E. h

uxle

yi (p

art.m

l-1)

Temps (jours)1 3 5 7 9 11 13

B

AVirus-induced mortality

From Jacquet et al. 2002

Ecological Role of Aquatic VirusesVirus-induced mortality

From Weinbauer & Hofle 1998From Simek et al. 2001

Ecological Role of Aquatic VirusesVirus-induced mortality

Viruses remove app.10 to 50% of prokaryotic biomass per day

Mortality by viruses equals grazing by small eukaryotic predators

Ecological Role of Aquatic VirusesViruses and organic carbon release

From Wommack & Colwell 2000

Ecological Role of Aquatic Viruses

Phytoplankton100% Grazers DOC

Heterotrophicbacteria

Carnivores

Viruses Viruses

Viruses

Recycling of6-26%

3-15%1%2-10%

From Wilhelm & Suttle 1999

Cell leakage <10%

17-35%

80-88%15-43%

18-52%3-9%

2-9%

Virus shunt

Viral-induced carbon release : 0.1-10 µgC L-1 d-1

Viruses and organic carbon release

Ecological Role of Aquatic Viruses

Phytoplankton100% Grazers DOC

Heterotrophicbacteria

Carnivores

Viruses Viruses

Viruses

Recycling of6-26%

3-15%1%2-10%

From Wilhelm & Suttle 1999

Cell leakage <10%

17-35%

80-88%15-43%

18-52%3-9%

2-9%

Virus shunt

Viral lysates can sustain up to 30 % of bacterial carbon demand

Viruses and organic carbon release

Ecological Role of Aquatic Viruses

Phytoplankton100% Grazers DOC

Heterotrophicbacteria

Carnivores

Viruses Viruses

Viruses

Recycling of6-26%

3-15%1%2-10%

From Wilhelm & Suttle 1999

Cell leakage <10%

17-35%

80-88%15-43%

18-52%3-9%

2-9%

Virus shunt

The virus shunt decreases the efficiency of carbon transfer to higher trophic levels

Viruses and organic carbon release

Ecological Role of Aquatic VirusesViruses and organic carbon release

From Suttle 2005

Faster rate of CO2 build-up in the atmosphereReduction of the biological pump efficiency

Ecological Role of Aquatic VirusesViruses and organic carbon release

It remains to be shown whether viruses have a stabilizing or destabilizing effect on

ecosystems or geochemical cycles

From Suttle 2005

Ecological Role of Aquatic Viruses

From Weinbauer & Rassoulzadegan 2004

Viral impact on biodiversity

Viral regulation of host community diversity

Ecological Role of Aquatic VirusesEcological Role of Aquatic Viruses

From Wommack & Colwell 2000

The ”killing the winner populations” process means that lytic viruses can keep in check competetive dominants andalow for the co-existance of less competetive populations

Viral impact on biodiversity

Ecological Role of Aquatic VirusesViral impact on biodiversity

From Bouvier & del Giorgio 2007

Viruses influence bacterial community compositionLow density does not represent a refuge against VIM

Ecological Role of Aquatic Viruses

Lysogenic conversion, including a phenotypic change of an infected bacteria is well known from medicine (e.g Diphteria, Scarlet fever)

Natural transduction

This process also takes place in aquatic habitats to give infectedcells some ecological selective advantage

ex : toxicity in some Cyanobacteria, Vibrio

Also, LGT has occurred several times so that viruses must be considered as major players in the evolution of cellular genomes

Almost nothing is known about that for aquatic habitats

With 109 bact/l, doubling time of 15-30 h, total volume of Zeu of3.7 x 107 km3, a genetic exchange event with a probability of 10-20would occur 106 times per day

Jiang et al (1998) Transfer rates of 3.7 x 10-8 transductants/UFPThis means 1014 transductions per year in the Tampa Bay

Ecological Role of Aquatic VirusesNatural transduction

Extrapolation suggests that marine phages transduce 1028 base pairs of DNA per year in the world’s ocean

Ecological Role of Aquatic VirusesNatural transduction

Horizontal gene transfer undoubtedly occurs in natural microbial communities. However, the scale of the

process, the benefit to hosts and viruses, and the implications for the evolution and genetic

structure of planktonic communities are poorly known

Perhaps the most interesting example of lateral gene transfer among phages and their hosts to date is the discovery of cyanophages that

contain homologs to genes that encode key components of the photosynthetic machinery found in cyanobacteria

Aquatic Viral Diversity

Morphology : mainly dsDNA tailed phages

Size : 25-400 [30-70] nm

Host range : several viruses (>10) for one prokaryotic host

Fingerprinting methods : PCR-DGGEPFGE : 4-630 [50-100] kb

From Weinbauer 2004

Metagenomics (= environmental community genomics)

Metagenomics has revealed high potential bacteriophagediversity with almost no homologues in the existing

databases (65 to 95%)

Phage therapy in aquatics

ISI Web of knowledge (all years) :

phage AND therapy AND aquaculture : 10 citations

bacteriophage AND therapy AND aquaculture : 9 citations

(bacterio)phage AND therapy AND marine OR ocean : 0 citation

Looking for a bio-control or bio-remediation ?

Phage therapy in aquaticsLooking for a biocontrol ?

From Safferman & Morris (1963)

Large amount of studies : the potential importance of cyanophages as controlling factors for cyanobacterial blooms

Cyanophages unlikely to be useful as biological control agents : ecological investigations largely ceased

Phage therapy in aquatics

Aquaculture industry : ~ 30% of the seafood for human consumption

Millions of tons of fishes (but also crustaceans and shells !)

Net value of billions of euros (or dollars) !

Phage therapy in aquatics

From Paterson et al. (1969) to Imbeault et al. (2006), a dozen of papers related to the application of bacteriophages to Aeromonas salmonicida fish pathogens

Wu & Chao (1982) : Effects of phages on Edwardsiella tardainfectious of loaches (Taïwan)

Park et al. (1997) and Nakai et al. (1999) : Effects of phages on Lactococcus garvieaea infectious of yellowtail (Japan)

Park et al. (1997-2000) : Effects of phages on Pseudomonas plecoglossicida infectious of ayu

Phage therapy in aquatics

Many results mainly obtained in the lab or in restricted and protected volumes (aquariums) not directly in the field !

Take Home MessagesInterest in the ability of phages to control bacterial populations has extended from medical applications into the fields of agriculture, aquaculture, the food industry, in wastewater treatment systems as one alternative method to antibiotics of treating aquatic diseases

There are a lot of examples dealing with the use or attempts of phage therapy for preventing and controlling bacterial infections in -- aquaculture (farming) with subsequent diseases in Fishes (trout, salmon, yellowtail, ayu) CoralsCrustaceans (Lobsters, shrimps)Mollusks (Oyster)- wastewater treatment processes

Active against many different pathogens (Flavobacter, Pseudomonas, Vibrio, Aeromonas, Lactococcus, Piscirickettsia, etc) instead of the use of antibiotics (oxytetracycline, fluroquinolone, florfenicol, sulfadimethoxine-ormetoprim, etc)

Huge viral reservoirand potential but...

Phage therapy in aquatics

Phage therapy is now been considered an alternate to antibiotics and is an eco-friendly approach to fish and shrimp health management

But…

Co-existence of viruses and hosts is possible Interactions can drive genetic diversity in host populationbecause of gene transfer:

- leading to lysogeny in pathogens- leading to induction of enhanced virulence

Phage therapy in aquatics

“Although the idea of viruses as biological agents is an appealing prospect, the diversity and complexities of

life in the sea must be considered carefully.

More knowledge and great caution is needed before we attempt to apply processes similar to those used in agricultural

biocontrol or land” (Munn 2006)

Phage therapy in aquatics

Take Home Messages

Viruses in aquatic ecosystems are a major cause of cellular

mortality, a driver of microbial diversification and

global geochemical cycles, and the reservoir of

the greatest genetic diversity on Earth

Aquatic viral ecology =

only 20 years of study !

Take Home Messages

Aquatic ecosystems = Huge and unknown reserve of biodiversity = Source of many potential applications for biotechnologies

Methodologies and concepts in ecology may serve new applications in the medical field

Techniques in Medicine may serve new applications in the aquatic field

“All the world is a phage”

William Shakespeare

Thank you