Microbial Communities

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Microbial Communities Feeding Specialization of
Insects at The Sevilleta LTER

• Dan Colman

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Focal Organism Epicauta longicollis

• Member of Meloidae family
• 18-25 mm in length, slender, elongated body form
• Found throughout desert southwest Mexico
• Emerge in early summer
• Occur in swarms, feed
• on varying flowering
• plants
• Predominantly feeds
• on Solanum elaeagnifolium
• at the Sevilleta LTER
• (Schafer Toolson, 2007)

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Solanum elaeagnifolium(silverleaf nightshade)

• Perennial weed native to the western U.S. S.
  America
• 0.3 - 0.9m in height
• Very low soil quality water requirements
  adapted to semi-arid
• environments
• Contain varying levels
• of alkaloid neurotoxin, solanine,
• throughout plant
• Problematic for ranchers
• 3-6 mg/kg body weight can lead
• to fatality
• Grazing is very minimal
• E. longicollis only known grazer
• at the Sevilleta LTER

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Previous Research On E. longicollis

• Behavioral studies recently conducted at the
  Sevilleta LTER by Schafer Toolson
• Investigation into the feeding behavior of E.
  longicollis
• Strong evidence seen for feeding preference on
  varying plant parts
• Specialization seen in feeding indicates
  adaptation to feed predominantly on a plant of
  known high toxicity

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Objectives For The Study

• Characterize the microbial community residing
  within E. longicollis
• Compare against microbial communities within
  other insects of same region (various feeding
  habits)
• Analyze microbial communities over time intervals
  for changes in structure
• Analyze intraspecific community variation
• Determine if microbial community growth on S.
  elaeagnifolium media mirrors E. longicollis
  feeding preference

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Hypotheses

• Hypotheses
• Specialized feeders like E. longicollis will
  have a more specialized microbial community than
  non-specialized feeders.
• Microbial growth quantities species richness
  on S. elaegnifolium media will mirror E.
  longicollis feeding preference
• Analysis between individuals of the same
  species will show less variability than analysis
  interspecifically

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Justification For Research

• To adequately understand natural history of
  insects an understanding of endemic microbial
  communities is necessary
• Contribute to the understanding of co-evolution
  between microbial communities their hosts
• E. longicollis specialized diet may indicate the
  presence of a novel microbial strain/organism in
  microbial community
• Add evidence for hypotheses
• about microbial dispersal
• among distinct island habitats

http//www.jgi.doe.gov/education/bioenergy/bioener
gy_4.html
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Environmental Sampling A genetics Based Approach

• Cultivation is inherently biased to a few types
  of microorganisms, often not the most important
• Useful as a supplement to a genetics based
  approach
• Estimated that a very small amount of bacteria
  are culturable
• The most effective technique available for
  sampling microbial communities
• Allows us to take a snapshot at a specific time
  of community

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Environmental Sampling A Genetics Based Approach

• Community Sample

Total Community DNA
Selection Amplification of Target Genes
Insertion of Target Genes Into Vector
Vectors Taken In By Live E. coli Cells
Data Analysis
E. coli Colonies Grown, Samples Selected at random
Each Cultures Plasmid (Vector) DNA Harvested
Sequenced
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Other Species Sampled

• Pasimachus elongatus

Omorgus scutellaris

• 9-15 mm in length
• Found in southern U.S. Mexico (arid areas)
• Associated with scat, particularly coyote owl
• Detritivores (fur, skin, feathers in scat)

• 21-28 mm in length
• Found from plains of S.W. north to Canada
• Feed on smaller invertebrates

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Other Species Sampled

• Trimerotropis pallidipennis

• 25-45 mm in length
• Found from British Columbia to Mexico
• Generalist feeder (grasses and forbs)

Eleodes longicollis

• - Common darkling beetle of the desert southwest
• Feed on bits of seeds vegetation (associated
  with Pogonomyrmex rugosus ants)
• Defensive secretions that give them the
  stinkbug pseudonym

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Other Species Sampled

• Cibolacris parviceps (cream grasshopper)

• 20 32 mm in length
• Found throughout the S.W. U.S. into northern
  Mexico
• Thrives in desert habitats
• Feeds on creosote bush

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Study Sites

• All insects sampled from two areas within close
  proximity (6 km) and of similar vegetative growth

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Sampling Methods

• Each species sampled from one of the two site
  locations
• 3 individuals initially sampled for all 5
  comparison species 1 individual captured every
  week for two weeks
• Epicauta sampling differed
• Samples cooled upon capture
• Frozen upon returning from the
• field

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Dissection/Preparation for DNA Extraction

• Dissections were performed using sterile
  technique
• Performed as soon as possible upon return from
  field
• Dissection protocol identical for every sample
• Entire intestinal tract prepared for sampling by
  adding to a phosphate buffer solution

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DNA Extraction

• Bead beating step first used to help bring sample
  tissue into a useable solution
• Chemical process employed using standard DNA
  extraction protocol
• Cells in solution lysed (CTAB)
• Free proteins digested
• (Proteinase K)
• Cell membranes pulled apart
• (SDS Detergent)
• DNA in solution separated by
• extracting DNA into distinct
• layer (Chloroform)
• DNA precipitated out of solution
• into a pellet form

Pure DNA
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Polymerase Chain Reaction

• Enables us to select for one gene amplify
  quantity of that gene found in solution
• Reaction targets 16s rRNA gene conserved across
  microbial lineages
• A reaction mixture is made
• with all the necessary reagents
• for target genes to be
• duplicated according to
• standard PCR protocol (primers,
• polymerase, nucleotides, sample
• DNA, reaction buffer)
• Universal primers used

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Polymerase Chain Reaction

• Reaction mixtures are first heated
• Allows DNA double strands to separate
• Then cooled Primers attach at target gene ends
• Reaction then slightly heated again - polymerase
  attaches
• Polymerase attaches free nucleotides to template
  at primer 3 end
• Gene duplicate copies are exponentially made with
  every full cycle (a total of 30 cycles)
  potentially many 16s rRNA genes representing many
  different microbes can be amplified hundreds of
  thousands of times
• PCR is a sensitive process troubleshooting
  often required to have a successful reaction

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Monitoring For Positive Results

• PCR products analyzed before moving forward
• PCR reaction products
• ran on agarose gels
• Gels analyzed for
• fluorescence at 1500-
• 1600 base pair level
• which indicates presence
• of 16s rRNA in reaction
• product
• Successful reactions
• saved for processing

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Inserting Target Genes Into Vectors

• Target genes must be isolated separated from
  one another
• Insert each target gene into a cloning vector
• Each copy obtained from PCR now isolated from one
  another
• Housed individually in vectors
• Reaction with PCR
• product vectors
• Upon incubating the reaction at room
• temperature, our target gene is taken
• in by the vector

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Vectors Taken Up By Live Escherichia coli

• E. coli possess the ability to uptake free DNA
  from environment
• Reaction promotes uptake of vectors (with target
  genes now inserted)
• Each E. coli cell represents one of our target
  genes
• Cells cultivated colonies formed
• Each colony will contain identical copies of
  vector within all cells of colony
• 96 colonies will be selected at random per
• each insect sample

http//strubiol.icr.ac.uk/extra/baculovirus/bact_p
rotocols.html
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Vector DNA Harvested From E. coli

• Plasmid DNA is harvested from colony samples
• Similar process to DNA extraction initially
  performed
• Each colonys DNA then sequenced
• All 96 samples sequences ran through National
  Center For Biotechnologys Database of known
  sequences
• Nucleotide sequence similarity/distance indicates
  relatedness to known species
• 96 sequences per insect sample now available for
  analysis

http//biosci.sierracollege.edu/resources/bio-teac
hing/pcr-id.html
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Environmental Sampling A Genetics Based Approach

• Community Sample

Total Community DNA
Selection Amplification of Target Genes
Insertion of Target Genes Into Vector
Vectors Taken In By Live E. coli Cells
Data Analysis
E. coli Colonies Grown, Samples Selected at random
Each Cultures Plasmid (Vector) DNA Harvested
Sequenced
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Genetic Sampling Supplement Cultivation
Experiment

• 6 teas were made using Solanum elaeagnifolium
  plant tissue
• 3 types of plant tissue 2 treatments
• Concentration of tissue/water constant for all 3
• Tea medium filter sterilized
• Inoculated with pool
• of Epicauta intestinal
• material
• Incubated for 9 days
• Samples to be
• genetically identified

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Preliminary Observations

• Preliminary microscopy
• All 3 medium types look similar
• Cell counts seem to be low
• Predominantly a few shapes seen
• Genetic sampling of all 6 treatments necessary
  for conclusions to be made

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Future Data Analysis

• Each sequence represents one data point (one
  microbe individual originally in sample)
• Rarefaction Curves with unique sequences
• Cluster Analysis Arrange sequences into
  phylogenetic trees (per each sample, all samples
  total, per each species, etc.)
• Significant differences in community structure
  can be analyzed between
• 1) Individuals of the same species
• 2) All species
• 3) Species of different feeding behavior
  (specialized/non-specialized)
• 4) Different collection time points

Mesak et al. BMC Microbiology 2004 4 6
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Predictions For Results

• There will be a significant difference in
  communities between specialized non-specialized
  feeders
• Intraspecific analysis will show less variability
  than analysis interspecifically
• Community analysis of growth in medium will show
  less diversity along a gradient with known
  solanine presence

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Acknowledgements

• Dr. Cristina Vesbach
• Dr. Eric Toolson
• Jennifer Johnson
• NSF UNM Sevilleta LTER
• Scott Collins, John Dewitt,
• Don Natvig
• U.S. Fish Wildlife
• All of the REUs who helped
• throughout the summer
• especially Emerson Tuttle,
• Ash Schafer, Damon Lowery,
• Ashley Melendez Emily Stinson.

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Questions?