This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.
AbstractTermites are among the few animals known to have the capacity to subsist solely by consuming wood. The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson & Breznak, 1983). Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host. The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi & Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment. The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi & Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).
Procedural summary for termite whole-gut DNA extraction:
- Chill termites on ice, remove gut using sterile tweezers and stabilize gut samples in buffer.
- Homogenize samples in PVPP/SDS/phenol buffer.
- Extract and purify DNA from crude lysate using Qiagen DNeasy columns.
- On ice, remove the guts from worker caste termites using sterile forceps.
- Immediately transfer the guts and contents to a sterile, nuclease-free tube containing 50 μL ice-cold 1x molecular biology grade TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). Freeze samples at -20°C, or proceed directly with homogenization.
- Transfer the gut samples and buffer to a sterile, nuclease-free 2 ml screw capped tube pre-loaded with 500 mg of sterile zirconia/silica beads (0.1 mm) and 700 μl of 1x TE buffer containing 1% w/v polyvinylpolypyrrolidone (PVPP).
- Add 50 μl of 20% Sodium dodecyl sulfate (SDS) and 500 μl of phenol to the samples.
- Homogenize (bead beat) on the highest setting using three cycles of 30 sec homogenization and 30 sec of chilling on ice.
- Sediment insoluble material for 1 min at 8,000x g.
- Purify 300-μl aliquots of the aqueous (uppermost) layer with Qiagen DNeasy columns using the method described for crude lysate purification.
- Quantify nucleic acid content and freeze samples at -20°C for later use.
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In our experience, DNA extracted from the microbial communities of wood-feeding termite species like Zootermopis nevadensis is sufficiently pure for PCR template after one round of extraction and purification. However, some termites such as litter-feeding and soil-feeding species may have a higher concentration of humic acids in their gut contents and may require additional purification of gut microbial DNA. The total DNA yield from the guts of 5 Z. nevadensis workers is in the range of 10-30 μg. For termite species significantly smaller or larger than this species, more or fewer specimens may be needed to obtain a similar amount of DNA.
This method can easily be adapted to allow RNA extraction. For RNA extraction, substitute 1x RNAprotect Bacteria reagent from Qiagen (catlog no. 76506) for the ice-cold TE buffer described in step 2 of the protocol. Qiagen RNeasy reagents and columns (catlog no. 74104) should be used in place of the DNeasy purification procedure described above. As DNA can be purified from RNAprotect-stabilized samples and only 300 μL of the approx. 700 μL aqueous layer retrieved in step 7 is required for nucleic acid purification, this method can be used to retrieve both DNA and RNA in parallel from a single sample.
These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment. The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Moré et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi & Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).
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|DNeasy Tissue Kit||Kit||Qiagen||77607||Used according to the protocol for isolation of genomic DNA from crude lysates (Appendix H, product manual version: July 2003)|
|TE||Buffer||Sigma-Aldrich||T9285||1x buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) from 100x concentrate|
|zirconia/silica beads||Supplies||Biospec Products||11079101z||0.1 mm|
|PVPP||Reagent||Sigma-Aldrich||P6755||1% w/v polyvinylpolypyrrolidone prepared from dry reagent as a 1x suspension in TE buffer|
|SDS||Reagent||Sigma-Aldrich||L4390||Sodium dodecyl sulfate 20% soln. in water from dry reagent|
|BSS||Buffered Salt Solution, pH 7.2||Formulation per liter: 2.5 g K2HPO4, 1.0 g KH2PO4, 1.6 g KCl, 1.4 g NaCl, 0.075 g CaCl, 1 g MgCl, and 10 mL of a 1 M soln. of NaHCO3|
|AxioPlan-2||Microscope||Carl Zeiss, Inc.||Outfitted with 40x objective, 1.6x optivar and 10x ocular lenses. Samples were viewed using phase contrast illumination|
- Berthelet, M., Whyte, L. G., Greer, C. W. Rapid, Direct Extraction of DNA from Soils for PCR Analysis using Polyvinylpolypyrrolidone Spin Columns. FEMS Microbiol. Lett. 138, 17-22 (1996).
- Kudo, T., Ohkuma, M., Moriya, S., Noda, S., Ohtoko, K. Molecular Phylogenetic Identification of the Intestinal Anaerobic Microbial Community in the Hindgut of the Termite, Reticulitermes Speratus, without Cultivation. Extremophiles. 2, 155-161 (1998).
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- Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J. R. Microfluidic Digital PCR Enables Multigene Analysis of individual Environmental Bacteria. Science. 314, 1464-1467 (2006).
- Purdy, K. J., Embley, T. M., Takii, S., Newdell, D. B. Rapid Extraction of DNA and rRNA from Sediments by a Novel Hydroxyapatite Spin-Column Method. Appl. Environ. Microbiol. 62, 3905-3907 (1996).
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- Schmitt-Wagner, D., Friedrich, M. W., Wagner, B., Brune, A. Phylogenetic Diversity, Abundance, and Axial Distribution of Bacteria in the Intestinal Tract of Two Soil-Feeding Termites (Cubitermes spp). Appl. Environ. Microbiol. 69, 6007-6017 (2003).
- Tsai, Y. L., Olson, B. H. Rapid Method for Separation of Bacterial DNA from Humic Substances in Sediments for Polymerase Chain Reaction. Appl. Environ. Microbiol. 58, 2292-2295 (1992).