Microbial Communities & Feeding Specialization of Insects at The Sevilleta LTER

Dan Colman

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)

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 grazerat the Sevilleta LTER

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

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

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

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/bioenergy_4.html

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

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

E. coli Colonies Grown, Samples Selected at random

Each Culture’s Plasmid (Vector) DNA Harvested & Sequenced

Data Analysis

Other Species SampledPasimachus elongatus

- 21-28 mm in length

- Found from plains of S.W. north to Canada

- Feed on smaller invertebrates

-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)

Omorgus scutellaris

Other Species Sampled

Trimerotropis pallidipennis

-25-45 mm in length

- Found from British Columbia to Mexico

- Generalist feeder (grasses and forbs)

- 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

Eleodes longicollis

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

Study Sites

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

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

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

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 byextracting DNA into distinctlayer (Chloroform)

• DNA precipitated out of solutioninto a pellet form Pure DNA

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 tostandard PCR protocol (primers, polymerase, nucleotides, sampleDNA, reaction buffer)– Universal primers used

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

Monitoring For Positive Results• PCR products analyzed before moving forward

• PCR reaction productsran on agarose gels

• Gels analyzed forfluorescence at 1500-1600 base pair level which indicates presence of 16s rRNA in reaction product

• Successful reactions saved for processing

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

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_protocols.html

Vector DNA Harvested From E. coli

• Plasmid DNA is harvested from colony samples – Similar process to DNA extraction initially performed

• Each colony’s DNA then sequenced

• All 96 sample’s sequences ran through National Center For Biotechnology’s Database of known sequences– Nucleotide sequence similarity/distance indicates

relatedness to known species• 96 sequences per insect sample now available for

analysishttp://biosci.sierracollege.edu/resources/bio-teaching/pcr-id.html

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

E. coli Colonies Grown, Samples Selected at random

Each Culture’s Plasmid (Vector) DNA Harvested & Sequenced

Data Analysis

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 intestinalmaterial

• Incubated for 9 days• Samples to be

genetically identified

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

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 species2) All species3) Species of different feeding behavior (specialized/non-specialized)4) Different collection time points

Mesak et al. BMC Microbiology 2004 4: 6

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

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.

Questions?