The effects of preservatives and temperatures on arachnid DNA

VINK, C.; HEDIN, M.; PAQUIN, P.; HAYASHI, C.; ARNEDO, M.; BOND, J.; CODDINGTON, J.; GOLOBOFF, P. A.; GRISMADO, C. J.; GRISWOLD, C.; HORMIGA, G.; MADDISON, W.; MILLER, J.; PLATNICK, N. I.; PRENDINI, L.; RAMÍREZ, M. J.; SCHARFF, N.; SHEAR, W.; SIERWALD, P.; WHEELER, W.

Gent, Bélgica

Congreso; 16th International Congress of Arachnology; 2004

International Society of Arachnology

There have been a number of studies investigating the effects of different preservation techniques on DNA extraction in insects. Various claims have been made as to the minimum requirements for the preservation of useable arachnid DNA, however, empirical studies on the effect of preservatives and/or time on arachnid DNA have been limited.The usual method reported for the storage of spiders and other arachnids for DNA based studies has been in 95-100% ethanol with specimens either stored in a refrigerator or freezer. DNA degradation has been reported in spiders stored in 70-75% ethanol and short segments (<300 bp) of mitochondrial DNA have been amplified from spiders stored in 70% ethanol. However, longer segments of DNA (~1000 bp) are now more commonly used in DNA based phylogenetic studies and this requires DNA that has not been degraded by poor storage methods. The use of nuclear DNA is becoming more common in phylogenetic studies and are often present in low numbers, which also require good quality DNA.We tested the effects of different preservatives and temperatures on the yield of useable DNA in spider (Pardosa falcifera F.O. P.-Cambridge, 1902) and scorpion (Smeringurus mesaensis (Stahnke, 1957)) specimens.Six preservation treatments were used:1) Spiders and scorpions placed in 95% ethanol and left in 95% ethanol for six weeks.2) Spiders and scorpions placed in 70% ethanol and left in 70% ethanol for six weeks.3) Spiders and scorpions placed in 95% ethanol and transferred to 70% ethanol after 1 day and left in 70% ethanol for six weeks. This method may preserve the DNA and keep specimens suitable for morphological work.4) Spiders and scorpions placed in 70% ethanol and transferred to 95% ethanol after 1 week and left in 95% ethanol for five weeks. This is to simulate a collector not having 95% with them in the field.5) Spiders and scorpions placed in propylene glycol and left for six weeks, then transferred to 95% ethanol for one day at 4°C prior to extraction.6) Whole spiders, euthanized by freezing, were placed in 1 ml of RNAlater (as per manufacturer’s protocol. The patella, tibia, basitarsus, telotarsus and pretarsus of leg III of the scorpions were placed in 1 ml of RNAlater. Specimens were left in RNAlater for six weeks.