Rapd Profiling of Spider (araneae) Dna

We present protocols and conditions for specimen storage, DNA extraction and storage, and the subsequent RAPD (Random Amplified Polymorphic DNA) profiling of spiders . Three common UK species, Lepthyphantes tenuis (Blackwall 1852), Enoplognatha ovata (Clerk 1757) and Clubiona reclusa (Cambridge 1863), members of the Linyphiidae, Theridiidae and Clubionidae respectively, were chosen to serve as examples with this highly adaptable technique . Despite numerous reservations regarding the repeatability, homology, and statistical analysis of the data (see Grossberg et al . 1996 for a comprehensive review), RAPD profiling (Williams 1990) is still the method of choice for many researchers looking to address a wide range of ecological issues in an equally diverse array of organisms . RAPD data have enabled insights into population structure (e.g ., Haymer & McInnis 1994 ; Kambhampati et al . 1992), geographical origins and invasion routes of colonizing species (e .g ., Williams et al. 1994), the distinction of new genotypes of parasites (e.g ., Majiwa et al . 1993) and conservation genetics (e.g., Rosetto et al . 1995) . The RAPD technique can also be a useful initial step in detecting other classes of DNA marker such as microsatellites (Ender et al . 1996) . RAPD profiling is adopted despite the reservations because it possesses many advantages over other molecular marker systems, viz ., it is relatively fast and technically undemanding, screens the entire genome for polymorphisms, and can produce a potentially limitless number of markers (simply by screening with more primers). Moreover, due to the amplification process during the PCR thermal cycling, only minute quantities of DNA are required as template, making the analysis of invertebrates unproblematic, e.g ., microhymenoptera (Landry et al. 1993) . Sample storage prior to DNA extraction was found to be the most crucial stage for this otherwise robust technique, which worked successfully with all the species tested (Fig . 1). Spiders were collected via a D-Vac suction sampler, or by hand, and returned to the lab alive. They were then either stored in ethylene glycol or 70% ethanol at room temperature, or frozen in liquid nitrogen and stored at -80 °C . DNA was extracted after three weeks and examined on a 1% agarose minigel . RAPD reactions were then carried out with DNA stored at 4 °C and -20 °C over a period of one month, to assess the optimal storage for extracted DNA . Ethylene glycol and 70% ethanol were both found to be poor preservative media for the spider DNA, which had degraded substantially after three weeks storage at room temperature. Storage at -80 °C was found to be the most effective method tested for preserving specimens (at least for one year) prior to DNA extraction if extractions could not be made immediately (Fig . 2). However, it was necessary to identify the spiders prior to storage at -80 °C, as the delicate tissues of the epigyna and palps darkened following freezing, making identification more difficult . Saturated salt solutions have also been used by a number of authors as a means of preserving DNA during field collection of samples, e .g ., (Seutin et al . 1991) but these were not investigated in this study . Storage of extracted DNA at -20 °C is recommended if the sample is not to be used directly, as DNA held at 4 °C gave more variable results over time (results not shown) . Fresh dilutions of DNA should be prepared from -20 °C stock prior to each RAPD reaction to ensure repeatability of profiles (Fig . 3) . The DNA extraction was carried out as follows. A 1 .5 ml Eppendorf tube containing an adult spider was lowered into liquid nitrogen for 10 sec and the spider tipped out onto a Petri dish lid. The abdomen was removed with a sterile scalpel blade, preventing the possible