The effects of preservatives and temperatures on arachnid DNA
Cor J. Vink A C , Steven M. Thomas A , Pierre Paquin A , Cheryl Y. Hayashi B and Marshal Hedin AA Department of Biology, San Diego State University, San Diego, California 92182-4614, USA.
B Department of Biology, University of California, Riverside, California 92521-0427, USA.
C Corresponding author. Email: cor.vink@arachnology.org
Invertebrate Systematics 19(2) 99-104 https://doi.org/10.1071/IS04039
Submitted: 22 December 2004 Accepted: 12 April 2005 Published: 28 June 2005
Abstract
We tested the effects of different preservatives and temperatures on the yield of spider and scorpion DNA useable for PCR amplification. Our experiment was designed to simulate conditions in the field and laboratory over a six-week time period, testing the preservatives RNAlater®, propylene glycol, and various ethanol concentrations. Three replicates of each preservation treatment were stored at five different temperature treatments; –80°C, –20°C, 2–4°C, 19–24°C, and 40°C. DNA was extracted and quality was assessed by electrophoresis on mini-gels, and by PCR amplification of high copy mitochondrial DNA fragments (cytochrome oxidase subunit I) and low copy nuclear DNA fragments (actin). Results show that RNAlater® and propylene glycol are significantly better than the other preservatives for high quality DNA preservation and that tissue is best stored at –80°C or –20°C. Storage in 95% ethanol is appropriate if specimens are stored at –20°C or –80°C. We believe our results can help guide biologists in choosing preservatives and temperatures for DNA-based research on arachnids, other arthropods and invertebrates in general.
Additional keywords: DNA degradation, DNA preservation, PCR, scorpion, spider.
Acknowledgments
Marie Hudson, Jim Starrett, Ian Ballard and Debra Wytrykush provided valuable assistance with the DNA extractions. Daniel Palmer, Marie Hudson, Jim Starrett, Steve Foldi, Ian Ballard, Erica Dale, Peter Jensen and Stacie Jensen aided with specimen collection. Tom Prentice, Roger Farley, and Rick Vetter provided logistical help. Thanks to Lorenzo Prendini, Jonathan Coddington and Miquel Arnedo for valuable comments during the initial development of this work. We thank Martín Ramírez for his assessment of the morphological condition of spider specimens that had been stored in propylene glycol. Gustavo Hormiga and three anonymous referees provided helpful comments on the manuscript. This research was funded as part of the NSF supported Assembling the Tree of Life: Phylogeny of Spiders, grant number EAR0228699 to Ward Wheeler, Jonathan Coddington, Gustavo Hormiga, Lorenzo Prendini and Petra Sierwald (http://research.amnh.org/atol/files/).
A’Hara S.,
Harling R.,
McKinlay R. G., Topping C. J.
(1998) RAPD profiling of spider (Araneae) DNA. Journal of Arachnology 26, 397–400.
Arnedo M. A.,
Coddington J. A.,
Agnarsson I., Gillespie R. G.
(2004) From a comb to a tree: phylogenetic relationships of the comb-footed spiders (Araneae, Theridiidae) inferred from nuclear and mitochondrial genes. Molecular Phylogenetics and Evolution 31, 225–245.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Austin A. D., Dillon N.
(1997) Extraction and PCR of DNA from parasitoid wasps that have been chemically dried. Australian Journal of Entomology 36, 241–244.
Bond J. E.
(2004) Systematics of the Californian euctenizine spider genus Apomastus (Araneae: Mygalomorphae: Cyrtaucheniidae): the relationship between molecular and morphological taxonomy. Invertebrate Systematics 18, 361–376.
| Crossref | GoogleScholarGoogle Scholar |
Colgan D. J.,
Brown S.,
Major R. E.,
Christie F.,
Gray M. R., Cassis G.
(2002) Population genetics of wolf spiders of fragmented habitat in the wheat belt of New South Wales. Molecular Ecology 11, 2295–2305.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Dillon N.,
Austin A. D., Bartowsky E.
(1996) Comparison of preservation techniques for DNA extraction from hymenopterous insects. Insect Molecular Biology 5, 21–24.
| PubMed |
Gurdebeke S., Maelfait J.-P.
(2002) Pitfall trapping in population genetics studies: finding the right “solution”. The Journal of Arachnology 30, 255–261.
Hedin M. C.
(1997) Molecular phylogenetics at the population/species interface in cave spiders of the Southern Appalachians (Araneae: Nesticidae: Nesticus). Molecular Biology and Evolution 14, 309–324.
| PubMed |
Hedin M. C., Maddison W. P.
(2001) A combined molecular approach to phylogeny of the jumping spider subfamily Dendryphantinae (Araneae: Salticidae). Molecular Phylogenetics and Evolution 18, 386–403.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Hormiga G.,
Arnedo M. A., Gillespie R. G.
(2003) Speciation on a conveyor belt: sequential colonization of the Hawaiian Islands by Orsonwelles spiders (Araneae, Linyphiidae). Systematic Biology 52, 70–88.
| PubMed |
Maddison W. P., Hedin M. C.
(2003a) Jumping spider phylogeny (Araneae: Salticidae). Invertebrate Systematics 17, 529–549.
| Crossref | GoogleScholarGoogle Scholar |
Maddison W. P., Hedin M. C.
(2003b) Phylogeny of Habronattus jumping spiders (Araneae: Salticidae), with consideration of genital and courtship evolution. Systematic Entomology 28, 1–21.
| Crossref | GoogleScholarGoogle Scholar |
Mantel N.
(1967) The detection of disease clustering and a generalized regression approach. Cancer Research 27, 209–220.
| PubMed |
Post R. J.,
Flook P. K., Millest A. L.
(1993) Methods for the preservation of insects for DNA studies. Biochemical Systematics and Ecology 21, 85–92.
| Crossref | GoogleScholarGoogle Scholar |
Quicke D. L. J.,
Belshaw R., Lopez-Vaamonde C.
(1999) Preservation of hymenopteran specimens for subsequent molecular and morphological study. Zoologica Scripta 28, 261–267.
| Crossref | GoogleScholarGoogle Scholar |
Riess R. A.,
Schwert D. P., Ashworth A. C.
(1995) Field preservation of Coleoptera for molecular genetic analyses. Environmental Entomology 24, 716–719.
Rubink W. L.,
Murray K. D.,
Baum K. A., Pinto M. A.
(2003) Long term preservation of DNA from honey bees (Apis mellifera) collected in aerial pitfall traps. The Texas Journal of Science 55, 159–168.
Simon C.,
Frati F.,
Beckenbach A.,
Crespi B.,
Liu H., Flook P.
(1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annals of the Entomological Society of America 87, 651–701.
Vink C. J., Paterson A. M.
(2003) Combined molecular and morphological phylogenetic analyses of the New Zealand wolf spider genus Anoteropsis (Araneae: Lycosidae). Molecular Phylogenetics and Evolution 28, 576–587.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Vink C. J.,
Mitchell A. D., Paterson A. M.
(2002) A preliminary molecular analysis of phylogenetic relationships of Australasian wolf spider genera (Araneae: Lycosidae). The Journal of Arachnology 30, 227–237.
Wheeler W. C., Hayashi C. Y.
(1998) The phylogeny of the extant chelicerate orders. Cladistics 14, 173–192.
| Crossref | GoogleScholarGoogle Scholar |
