Published on February 20, 2014
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/
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
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)
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 (Roudnew et al., 2012) • Confined aquifer • Sampled at 79-84 m • 200 L
Enumeration Flow cytometry
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)
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)
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
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)
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)
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 (PCVAD) (www.octagon-services.co.uk) Maize streak virus (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
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
Acknowledgments Microbial Systems Lab member James Paterson Industry Partners:
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