Viruses

Bacteria

Flow cytometry and methods to count aquatic viruses and assess viral-induced mortality of bacteria

Personnic S1, Duhamel S1, Sime-Ngando T2, Domaizon I1 & Jacquet S1 (1) UMR CARRTEL, Equipe de Microbiologie Aquatique, Thonon & Bourget-du-lac, France (2) Laboratoire de Biologie des Protistes, Campus des Cézeaux ,Aubière, France

Content Viruses: who are you ? The aquatic microbial network Viral roles Methods to count aquatic viruses EFM TEM FCM Comparison EFM / TEM / FCM Case studies dealing with the viral community dynamics Cases studies dealing with the viral role as mortality agents Conclusion

The aquatic viruses

• Are you numerous ? -10 to 100 more abundant than heterotrophic bacteria - About 105 to 108 particles per ml of water, the most abundant aquatic biological entities - Abundances: Viruses > Heter. Bacteria > Cyanobacteria > Pico-Nano eukaryotes

• Who are you ? - Size: 20 to 200 nm (most < 60 nm) - Diversity ? Poorly known - Mortality agents, obligatory parasites

Fig: Schema of a virus T4

- Ds DNA containing particles

• Are you fundamental to understand microbial ecology ?

The aquatic food web DOM allochtonous

Microbial loop

Phytoplankton

DOM

Heterotrophic Bacteria

Zooplankton

Protistes flagellates Higher Predators

Protistes ciliates

Viruses

DOM allochtonous DOM allochtone

Phytoplankton Phytoplankton

Microbial loop

DOM DOM

Heterotrophic Bacteria Heterotrophic Bacteria

Viruses Zooplankton Zooplankton Protistes flagellés Viruses Higher Higher Predators Prédators

Protistes Protistes ciliés flagellates

Protistes ciliates

The role of viruses

! Regulating factor of microbial communities abundance ; ! Regulating factor of bacterial diversity ; ! Responsible for bacterial gene transfer (transduction) .

⇓ . Understanding relations between microbial communities without viruses Not possible

. Analysing and explaining bacterial diversity without viruses ? Not possible

Need to analyze viral communities

Methods to count aquatic viruses

There are 3 principal techniques to count viruses in the field of aquatic sciences

. EFM EpiFluorescence Microscopy . TEM Transmission Electron Microscopy . FCM Flow Cytometry

Note that other techniques exist to enumerate viruses

EFM: Epifluorescence microscopy

Viruses

Bacteria Type: Microscopy Principle: Target bacterial and viral DNA fluorescence after light excitation. DNA fluorescence is obtained using a highly fluorescent acid-nucleic dye (SYBR Gold for example). Counts: human-eye

Advantages

Disadvantages Human counts (reproducibility ?)

See (pretend to see) the organisms Relatively quick method

Particle Fluorescence (fading) Conversion of the viruses really counted to viruses per ml

TEM: Transmission electron microscopy

Viruses Bacteria

Type: Microscopy Principle: Diffraction of an electron flow by colored bacterial and viral cell compounds Counts: human-eye

Advantages

Disadvantages Human counts (reproducibility ?)

See the organisms Access of different parameters: counts but also BS, FVIC (+FIC, VIBM)

Impossible to use it for routine quantification + skilled personnel Conversion of the viruses really counted to viruses per ml

BS: burst size, the numbers of viruses liberated by lytic events FVIC: the % of bacterial cells visibly infected by viruses FIC: the % of bacterial cells infected VIBM: the % of bacterial production removal due to viral lysis

FCM: Flow cytometry

Viruses

Bacteria

Type: Cytometry Principle: Target Bacterial and viral DNA fluorescence after laser excitation. DNA fluorescence is obtained with a highly fluorescent acid nucleic dye (SYBR Green I for example). Counts: device counts and software analysis

Advantages

Quick method Useful for routine quantification Reproducible

Disadvantages Do not directly see the organisms Over-estimation of viruses ? Particle size and fluorescence: limit of cytometry detection

Comparison EFM / TEM / FCM Bettarel Y., T. Sime-Ngando, C. Amblard and H. Laveran (2000). A comparison of methods for counting viruses in aquatic systems. Applied and Environmental Microbiology 66(6): 2283-2289.

6.E+06

1:1 FCM counts

Marie D., C. P. D. Brussaard, R. Thyrhaug, G. Bratbak and D. Vaulot (1999). Enumeration of marine viruses in culture and natural samples by flow cytometry. Applied and Environmental Microbiology 65(1): 45-52.

4.E+06

2.E+06

Weimbauer M.G., and T. Suttle. (1997). Comparison of epifluorescence and transmission electron microscopy for counting viruses in natural marine waters. Aquatic Microbial Ecology 13: 225-232.

0.E+00 0.00E+00

2.00E+06

4.00E+06

6.00E+06

EFM Counts

Virus-like particles counts can be significantly different ⇓ Difficulty to compare results obtained with different methods

Accessing viral dynamics Annecy

B o u rg e t

B

A

10

Depth (m)

G eneva

Flow cytometry

C

20

30 1e +2 1e +3 1e +4 1e +5 1e +6

40 -1

P ic o c y a n o ( c e ll.m l )

picoyanobacteria

50

Depth (m)

E

D

10

F

20

30

small eukaryotes 1e +2 1e +3 1e +4 1e +5 1e +6

40 -1

S m a ll e u k a r y o t e s ( c e ll.m l ) 50

G

Depth (m)

10

I

H

20

heterotrophic bacteria

30 1e+6 2e+6 3e+6 4e+6 5e+6

40 -1

H e t . b a c t e r ia ( c e ll.m l ) 50

J Data not available

Depth (m)

20

L

K

10

30

viruses

40

5e+7 1e+8 1 .5 e + 8 2e+8

-1

V ir u s ( p a r t .m l ) 50 Aug

Dec

2002

Apr

Aug

2003

D ec Aug

Dec

2002

Apr

Aug

2003

Dec

Aug

2002

Dec

Apr

Aug

2003

Dec

Accessing viral-induced bacterial mortality

Agents of bacterial mortality

Microcosms . DialysisBags . Bottles Exp. Enrichment Exp. Dilution

% of bacterial mortality due to the viral lytic activity

Direct ?

TEM

Not Direct ?

Dilution technique (FCM counts)

Accessing virus-induced bacterial mortality

Lake water ultrafiltrate (0.02 µm)

Grazer free Lake water 0% Control

20%

40%

70%

100%

Bacterial Growth rate (d-1)

Illustration of the dilution results y = -0,0045x + 0,8948 2 R = 0,9397 0.8

Dilution technique 0.6

0.4

0.2 0

20

40

60

80

100

Dilution

Contact rates bacteria/viruses

120

Accessing viral-induced bacterial mortality

Viral lyses (TEM)

Viral lyses (Dilution/FCM)

Flagellates Grazing

EXP. Lake Geneva May 2004

0-5 %.d-1

9 %.d-1

32 %.d-1

EXP. Lake Le Bourget April 2003

14 %.d-1

38 %.d-1

56 %.d-1

EXP. Lake Le Bourget May 2003

20 %.d-1

26 %.d-1

63 %.d-1

EXP. Lake Le Bourget August 2003

10 %.d-1

34 %.d-1

18 %.d-1

Heterotrophic Bacterial Mortality

Conclusion

Techniques for viral counts may give different results Techniques to asses viral-induced bacterial mortality may also give different results ⇓ We have to keep this in mind before making reliable comparisons between ecosystems ---Hence, the question is asked about the possibility of a common procedure or any universal way of counting and assessing mortality processes A future objective: European data basis / inter-calibration of methods ?

No evident answer yet and will be there any ?

1st European Workshop on Aquatic Phage Ecology Castle of Ripaille, Thonon-les-Bains, 2-4 February 2005

Abstract of the proposed workshop topic: Viruses infecting bacteria and/or cyanobacteria are an essential biological compartment in aquatic microbial food webs. They are important controlling agents for planktonic communities, playing a key role in cell mortality, nutrient cycles, microbial diversification and diversity. However, still little is known on how this viral activity is linked to diversity and ecosystem functioning. This first workshop in Europe will provide an opportunity for promoting this field of research through discussion and presentation of our past and most recent results, of the various approaches and methods we use and the problems we face, for reinforcing or developing collaboration, and in fine for developing strategies for future research.

Organization: Stéphan JACQUET INRA