Analyzing Arthropods for the Presence of Bacteria

Elizabeth S. Andrews1

1 Department of Entomology, University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1E.6
DOI:  10.1002/9780471729259.mc01e06s28
Online Posting Date:  February, 2013
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Abstract

Bacteria within arthropods can be identified using culture‐independent methods. This unit describes protocols for surface sterilization of arthropods, DNA extraction of whole bodies and tissues, touchdown PCR amplification using 16S rDNA general bacteria primers, and profiling the bacterial community using denaturing gradient gel electrophoresis. Curr. Protoc. Microbiol. 28:1E.6.1‐1E.6.14. © 2013 by John Wiley & Sons, Inc.

Keywords: arthropod; bacteria; community; sterilization; 16S rDNA; denaturing gradient gel electrophoresis; DGGE

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Bacterial DNA from Arthropod Tissue Samples
  • Alternate Protocol 1: Homogenization of Arthropods with Hard Bodies
  • Basic Protocol 2: Using PCR to Amplify Bacterial DNA from Arthropods
  • Basic Protocol 3: DGGE Analysis of PCR Products Using a Bio‐Rad DCode System
  • Support Protocol 1: Creation of a Ladder from Bacteria Bands
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Bacterial DNA from Arthropod Tissue Samples

  Materials
  • Arthropods or eggs for extraction
  • 70% ethanol
  • Molecular grade water, autoclaved
  • Phosphate‐buffered saline containing potassium (KPBS; appendix 2A), pH 7.4, autoclaved
  • Homogenization buffer (see recipe)
  • Qiagen DNeasy blood and tissue kit
  • Sterile pipet tips and pipetting aid
  • Forceps and dissecting tools, sterile
  • Glass microscope slides, sterile
  • 1‐mm glass beads, autoclaved
  • Bead beater or pestles for 1.5‐ml microcentrifuge tubes

Alternate Protocol 1: Homogenization of Arthropods with Hard Bodies

  Additional Materials
  • Liquid nitrogen

Basic Protocol 2: Using PCR to Amplify Bacterial DNA from Arthropods

  Materials
  • Molecular‐grade H 2O, autoclaved
  • 10× PCR buffer containing MgCl 2
  • 10 mM dNTPs
  • 20 µM forward primer: 5′‐CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG‐3′
  • 20 µM reverse primer: 5′ ‐ATT ACC GCG GCT GCT GG‐3′
  • Taq DNA polymerase (5 U/µl)
  • Template DNA ( protocol 1)
  • 1.5% agarose gel containing Gel Red (see recipe)
  • 0.5× TBE buffer (see recipe)
  • 1‐kb DNA ladder
  • 0.5‐ml thin‐walled PCR tubes
  • Automated thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ).

Basic Protocol 3: DGGE Analysis of PCR Products Using a Bio‐Rad DCode System

  Materials
  • 10% polyacrylamide denaturing solution (see reciperecipes)
    • Low concentration—40%
    • High concentration—70%
  • 10% ammonium persulfate (see recipe)
  • TEMED
  • Double‐distilled (dd) H 2O
  • 1× TAE run buffer (see recipe)
  • 2× gel‐loading dye (see recipe)
  • GC‐clamped PCR product ( protocol 3)
  • Gel Red solution (see recipe)
  • Molecular‐grade H 2O
  • 1.5% agarose gel containing Gel Red (see recipe)
  • Sequencing primer: 5′‐CCT ACG GGA GGC AGC AG‐3′
  • Bio‐Rad DCode universal mutation detection system
    • 16 × 16‐mm plates
    • 1‐mm spacers
    • Comb, 1 mm
    • Electrophoresis tank, temperature control module, and power supply
  • Variable flow mini‐peristaltic pump (Fisher Scientific, Model 3386‐medium flow)
  • Silastic laboratory tubing (Dow Corning, 1.57 mm I.D. × 2.18 mm O.D.)
  • Gradient mixer, 100 ml (Fisher Scientific, cat. no. 29‐237‐82)
  • Gel‐loading tips (Fisherbrand Premium Multiflex) 0.5‐200 µl
  • Paper towels or card stock
  • 10‐ml syringe, no needle
  • Staining container
  • Aluminum foil
  • Clinical rotator (Fisher Scientific)
  • UV transilluminator
  • Micro‐spatulas, metal, sterilized with flame
  • 0.5‐ml microcentrifuge tubes
  • Geneious Pro or other bioinformatics software
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ).

Support Protocol 1: Creation of a Ladder from Bacteria Bands

  Materials
  • See Basic Protocols protocol 32 and protocol 43.
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Figures

Videos

Literature Cited

Literature Cited
   Behar, A., Yuval, B., and Jurkevitch, E. 2008. Community structure of the Mediterranean fruit fly microbiota: Seasonal and spatial sources of variation. Isr. J. Ecol. Evol. 54:181‐191.
   Crotti, E., Balloi, A., Hamdi, C., Sansonno, L., Marzorati, M., Gonella, E., Favia, G., Cherif, A., Bandi, C., Alma, A., and Daffonchio, D. 2012. Microbial symbionts: A resource for the management of insect‐related problems. Microbial Biotechnol. 5:307‐317.
   Dale, C. and Moran, N.A. 2006. Molecular interactions between bacterial symbionts and their hosts. Cell 126:453‐465.
   Dillon, R.J. and Dillon, V.M. 2004. The gut bacteria of insects: Nonpathogenic interactions. Annu. Rev. Entomol. 49:71‐92.
   Dillon, R.J., Webster, G., Weightman, A.J., and Charnley, A.K. 2010. Diversity of gut microbiota increases with aging and starvation in the desert locust. Anton. Leeuw. Int. J. G. 97:69‐77.
   Fall, S., Hamelin, J., Ndiaye, F., Assigbetse, K., Aragno, M., Chotte, J.L., and Brauman, A. 2007. Differences between bacterial communities in the gut of a soil‐feeding termite (Cubitermes niokoloensis) and its mounds. Appl. Environ. Microbiol. 73:5199‐5208.
   Hui, X.A., Wei, G.F., Jia, S., Huang, J., Miao, X.‐X., Zhou, Z., Zhao, L.‐P., and Huang, Y.‐P. 2006. Microbial communities in the larval midgut of laboratory and field populations of cotton bollworm (Helicoverpa armigera). Can. J. Microbiol. 52:1085‐1092.
   Ishikawa, H. 2003. Insect symbiosis: An introduction. In Insect Symbiosis (K. Bourtzis and T.A. Miller, eds.) pp. 1‐22. CRC Press, Boca Raton, Fla.
   Kikuchi, Y. 2009. Endosymbiotic bacteria in insects: Their diversity and culturability. Microbes Environ. 24:195‐204.
   Lindh, J.M. and Lehane, M.J. 2011. The tsetse fly Glossina fuscipes fuscipes (Diptera: Glossina) harbours a surprising diversity of bacteria other than symbionts. Anton. Leeuw. Int. J. G. 99:711‐720.
   Moran, N.A. 2001. Bacterial menageries inside insects. Proc. Natl. Acad. Sci. U.S.A. 98:1338‐1340.
   Mrázek, J, Strosová, L, Fliegerová, K, Kott, T, and Kopecný, J. 2008. Diversity of insect intestinal microflora. Folia Microbiol. 53:229‐233.
   Muyzer, G., de Waal, E.C., and Uitterlinden, A.G. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction‐amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695‐700.
   Muyzer, G. and Smalla, K. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Anton. Leeuw. Int. J. G. 73:127‐141.
   Polz, M.F. and Cavanaugh, C.M. 1998. Bias in template‐to‐product ratios in multitemplate PCR. Appl. Environ. Microbiol. 64:3724‐3730.
   Reeson, A.F., Jankovic, T., Kasper, M.L., Rogers, S., and Austin, A.D. 2003. Application of 16S rDNA‐DGGE to examine the microbial ecology associated with a social wasp Vespula germanica. Insect Mol. Biol. 12:85‐91.
   Schabereiter‐Gurtner, C., Lubitz, W., and Rölleke, S. 2003. Application of broad‐range 16S rRNA PCR amplification and DGGE fingerprinting for detection of tick‐infecting bacteria. J. Microbiol. Methods 52:251‐260.
   Shi, W.B., Syrenne, R., Sun, J.‐Z., and Yuan, J.S. 2010. Molecular approaches to study the insect gut symbiotic microbiota at the ′omics' age. Insect Sci. 17:199‐219.
   Suzuki, M.T. and Giovannoni, S.J. 1996. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62:625‐630.
   Voytas, D. 2001. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Zahner, V., Lucarotti, C.J., and McIntosh, D. 2008. Application of 16S rDNA‐DGGE and plate culture to characterization of bacterial communities associated with the sawfly, Acantholyda erythrocephala (Hymenoptera: Pamphiliidae). Curr. Microbiol. 57:564‐569.
   Zhang, H. and Jackson, T.A. 2008. Autochthonous bacterial flora indicated by PCR‐DGGE of 16S rRNA gene fragments from the alimentary tract of Costelytra zealandica (Coleoptera: Scarabaeidae). J. Appl. Microbiol. 105:1277‐1285.
   Zouache, K., Raharimalala, F.N., Raquin, V., Tran‐Van, V., Raveloson, L.H., Ravelonandro, P., and Mavingui, P. 2011. Bacterial diversity of field‐caught mosquitoes, Aedes albopictus and Aedes aegypti, from different geographic regions of Madagascar. FEMS Microbiol. Ecol. 75:377‐389.
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