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/

What happens with reduction or removal of invertebrate grazers?

protistan predators?and bacteria?

Viruses remaineasy in the labimpossible in surficial aquatic ecosystemsit can happen in groundwater

Simple question

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)

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

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)

Groundwater structure

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

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 aquifer

• exposed to external input

• Confined aquifer

• approx. 1500 years old => baseline

Viral communities in confined aquifer

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

Field Methods

• Bores purged 3 bore volumes

• Unconfined aquifer• Sampled at 13-19 m• 20 L

• Confined aquifer• Sampled at 79-84 m• 200 L

(Roudnew et al., 2012)

EnumerationFlow cytometry

IdentificationMolecular

• 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)

IdentificationAnalysis

• 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)

ResultsGeophysical 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

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)

A B C

D E F

    100

101

102

103

104

101

102

103

104

101 102 103 104100

100 101 102 103 104100 101 102 103 104100

Side scatter (cell size)

SY

BR

GF

(nu

clei

c ac

id c

onte

nt)

Aquifer cytograms

(Roudnew et al., 2012)

1000 10000 100000 1000000

-105

-90

-75

-60

-45

-30

-15

0

Un-purged

Log bacteria ( ×105 cells mL-1)

Dep

th (m

)

1000 10000 100000 1000000Log VLP ( ×105 particles mL-1)

A B

Variation in groundwater abundances

Taxonomy

• ssDNA viruses (72%)

• dsDNA viruses (1%)

MEGAN

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

ssDNA viruses

• Circoviridae

Maize streak virusBeak and feather disease

Porcine Circovirus Associated Diseases

(PCVAD)

(www.environment.gov.au) (www.octagon-services.co.uk) (www.geminiviridae.wordpress.com)

MEGAN

Normalized Goodall’s similarity matrix

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

MEGAN

Further Circoviridae analysis

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

Conclusions

• Confined aquifers have unique circoviruses• Unknown endogenous source or

• Temporally and spatially distant large animal sources

AcknowledgmentsMicrobial 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 leukaemia virus paradox

Thankyou!