Venerable viromes
in cryptic, isolated and enduring ecosystems

Can viromes be archaeological microbial ecology?

Jim Mitc...
Simple question
What happens with reduction or removal of
invertebrate grazers?
protistan predators?
and bacteria?

Viruse...
Groundwater Importance
•

97% of the world’s unfrozen freshwater reserves

(Gibert &

Deharveng 2002)

•

2 billion people...
Biological Importance
• Shifts in the structure of microbial

assemblages related to human impact
(Dinsdale et al., 2008a)...
Microbial Importance
• Subsurface microbial communities represent the
greatest reservoir of micro-organisms (Whitman et al...
Groundwater structure
Confined Aquifers
• Confined aquifers are protected from
external input by aquitards (Nolan et al., 1997)
Inhibits contami...
Broad Aim

•

Determine microbial community structure in groundwater
Site
• Ashbourne
Site

• Under a dairy farming region
• Two aquifer flow systems – separate water
sources (Banks et al., 2007)
• Unconfined...
Viral communities in confined aquifer
Viral Risks
•

Viral pathogens compromising freshwater reserves globally
(Abbaszadegan et al., 2003)

•

Longer infectious...
Field Methods
• Bores purged 3 bore volumes
• Unconfined aquifer
• Sampled at 13-19 m
• 20 L
(Roudnew et al., 2012)

• Con...
Enumeration
Flow cytometry
Identification
Molecular

• MoBio PowerWater® DNA Extraction
• GS-FLX Pyrosequencing (Titanium) Sequenced

• MetaGenomics ...
Identification
Analysis

• Unassembled DNA sequence annotation
•

Community Cyberinfrastructure for Advanced
Microbial Eco...
Results
Geophysical data

• Characterised by low oxygen levels
• Approx. 0.2 mg L-1
• Statistically significant increase i...
Aquifer cytograms
104

A

B

C

D

E

F

103

SYBR GF (nucleic acid content)

102
101
100
104
103
102

101
100

100

101

...
Variation in groundwater abundances
Log bacteria ( ×105 cells mL-1)
0.01
0
-15

0.1
Unpurged
Purged

1

Log VLP ( ×105 par...
Taxonomy
•

ssDNA viruses (72%)

•

dsDNA viruses (1%)
MEGAN

• Circoviridae ,Geminiviridae & Nanoviridae
• Plant and animal pathogens
• Livestock diseases
• 17- 30 nm
ssDNA viruses
• Circoviridae

Beak and feather
disease
(www.environment.gov.au)

Porcine Circovirus
Associated Diseases
(P...
MEGAN
Normalized
Goodall’s
similarity
matrix
MEGAN
• Viral metagenome
• Despite geographical proximity, confined
aquifer more similar to reclaimed water
•

Viruses int...
MEGAN
Further Circoviridae analysis
Summary: ssDNA Viruses
• Small size
•

Greatest potential for transport

• High substitution rate
•
•

High pathogenicity
...
Conclusions
• Confined aquifers have unique circoviruses
• Unknown endogenous source or
• Temporally and spatially distant...
Acknowledgments
Microbial Systems Lab member
James Paterson
Industry Partners:
Discussion points

Archaeology and Tracking: long bow or basic principle?
Single site focus vs 500+ sites
2.2 kb Chicken l...
Thankyou!
Venerable Viromes In Cryptic, Isolated and Enduring Ecosystems - Jim Mitchell
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Venerable Viromes In Cryptic, Isolated and Enduring Ecosystems - Jim Mitchell

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Pore water microbial communities change as they move below the surface. The continuing isolation and evolution of a groundwater community may continue for hundreds of thousands of years. There is little known regarding the viral diversity and abundance of deep groundwater communities. Recent work in 1500 year old, 80 m deep, confined-aquifer groundwater showed unexpected viral groups to be abundant. Specifically, genomes of the large animal virus family Circoviridaewere were diverse and readily identifiable. These viruses have been found in human faeces, but whether they are human pathogens is still unknown. However, they are known to cause disease in most major livestock groups. Geminiviridaeand Microviridae were also present. These results demonstrate that viruses can spread and endure in groundwater as common members of the microbial community. The extent of the spread and endurance, as well as the extent to which the porous matrices of aquifers act to concentrate viruses is the focus on ongoing work. The results also raise the question of the extent to which relic viruses of some groundwater communities are a record of venerable if not quite ancient surface fauna and flora.

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Venerable Viromes In Cryptic, Isolated and Enduring Ecosystems - Jim Mitchell

  1. 1. Venerable viromes in cryptic, isolated and enduring ecosystems Can viromes be archaeological microbial ecology? Jim Mitchell, Renee Smith, Shane Tobe, Tom Jeffries, Ben Roudnew, Justin Seymour, Alison Fitch, Keryn Simons, Peter Speck, Kelly Newton, Melissa Brown GC Myers, http://redtreetimes.com/2011/11/30/archaeology-rainbows-end/
  2. 2. Simple question What happens with reduction or removal of invertebrate grazers? protistan predators? and bacteria? Viruses remain easy in the lab impossible in surficial aquatic ecosystems it can happen in groundwater
  3. 3. Groundwater Importance • 97% of the world’s unfrozen freshwater reserves (Gibert & Deharveng 2002) • 2 billion people rely on freshwater reserves for potable drinking water + industrial and agricultural production (Kemper 2004) • Important in Australia -> drought events (Mpelasoka et al., 2008)
  4. 4. Biological Importance • Shifts in the structure of microbial assemblages related to human impact (Dinsdale et al., 2008a) • The effect of human modification of groundwater ecosystems is poorly known (NOT GDEs) • Today’s proposal Some groundwater records ecosystem information for up to 106 yrs via viruses
  5. 5. Microbial Importance • Subsurface microbial communities represent the greatest reservoir of micro-organisms (Whitman et al., 1998) • Terrestrial Subsurface – 1030 • Form the base of the subterranean food web (Sherr & Sherr 1991) • Primary purifiers of groundwater (Danielopol et al., 2003)
  6. 6. Groundwater structure
  7. 7. Confined Aquifers • Confined aquifers are protected from external input by aquitards (Nolan et al., 1997) Inhibits contamination • Vertical fractures -> preferential pathways for the introduction of surface contaminants including microbial pathogens • Enteric viruses have the great potential for spread into deep aquifers (Eaton et al., 2007) 27 – 75 nm
  8. 8. Broad Aim • Determine microbial community structure in groundwater
  9. 9. Site • Ashbourne
  10. 10. Site • Under a dairy farming region • Two aquifer flow systems – separate water sources (Banks et al., 2007) • Unconfined aquifer • exposed to external input • Confined aquifer • approx. 1500 years old => baseline
  11. 11. Viral communities in confined aquifer
  12. 12. Viral Risks • Viral pathogens compromising freshwater reserves globally (Abbaszadegan et al., 2003) • Longer infectious periods (Yates et al., 1985; Diels, 2005) • Low light • Low temperatures • Potential spread of pathogenic viruses in groundwater
  13. 13. Field Methods • Bores purged 3 bore volumes • Unconfined aquifer • Sampled at 13-19 m • 20 L (Roudnew et al., 2012) • Confined aquifer • Sampled at 79-84 m • 200 L
  14. 14. Enumeration Flow cytometry
  15. 15. Identification Molecular • MoBio PowerWater® DNA Extraction • GS-FLX Pyrosequencing (Titanium) Sequenced • MetaGenomics Rapid Annotation using Subsystem Technology (MG-RAST) pipeline version 2.0 (Meyer et al., 2008)
  16. 16. Identification Analysis • Unassembled DNA sequence annotation • Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) (Seshadri et al., 2007) • Newly constructed database visualised in MEtaGenome Analyzer (MEGAN) (Huson et al., 2007)
  17. 17. Results Geophysical data • Characterised by low oxygen levels • Approx. 0.2 mg L-1 • Statistically significant increase in the unconfined compared to confined of: • Iron • Sulphur • Total Organic Carbon
  18. 18. Aquifer cytograms 104 A B C D E F 103 SYBR GF (nucleic acid content) 102 101 100 104 103 102 101 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Side scatter (cell size) Cytograms of groundwater samples from the unconfined aquifer (A), the confining layer (B) and the confined aquifer (C, D, E and F). (Roudnew et al., 2013)
  19. 19. Variation in groundwater abundances Log bacteria ( ×105 cells mL-1) 0.01 0 -15 0.1 Unpurged Purged 1 Log VLP ( ×105 particles mL-1) 10 A 0.01 0.1 1 10 B Depth (m) -30 -45 -60 -75 -90 -105 (Roudnew et al., 2012)
  20. 20. Taxonomy • ssDNA viruses (72%) • dsDNA viruses (1%)
  21. 21. MEGAN • Circoviridae ,Geminiviridae & Nanoviridae • Plant and animal pathogens • Livestock diseases • 17- 30 nm
  22. 22. ssDNA viruses • Circoviridae Beak and feather disease (www.environment.gov.au) Porcine Circovirus Associated Diseases (PCVAD) (www.octagon-services.co.uk) Maize streak virus (www.geminiviridae.wordpress.com)
  23. 23. MEGAN Normalized Goodall’s similarity matrix
  24. 24. MEGAN • Viral metagenome • Despite geographical proximity, confined aquifer more similar to reclaimed water • Viruses introduced prior to underlying proximity -> long term survival of viruses in groundwater
  25. 25. MEGAN
  26. 26. Further Circoviridae analysis
  27. 27. Summary: ssDNA Viruses • Small size • Greatest potential for transport • High substitution rate • • High pathogenicity Inherent stability • 1500 year old groundwater • Long term survival and spread of pathogenic viruses in groundwater ecosystem
  28. 28. Conclusions • Confined aquifers have unique circoviruses • Unknown endogenous source or • Temporally and spatially distant large animal sources
  29. 29. Acknowledgments Microbial Systems Lab member James Paterson Industry Partners:
  30. 30. Discussion points Archaeology and Tracking: long bow or basic principle? Single site focus vs 500+ sites 2.2 kb Chicken leukaemia virus paradox
  31. 31. Thankyou!
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