DNA extraction from spider webs

Many spider species produce webs that represent a potential non-invasive source of DNA for conservation genetic analysis. Reported here is the successful isolation of target DNA from members of two families (Theraphosidae and Pholcidae) using a standard CTAB phenol–chloroform–isoamyl protocol. The isolated DNA was of sufficient quality to permit routine PCR amplification and sequencing of mtDNA COI fragments of various sizes (maximum 710 bp attempted). This adds to other studies in demonstrating that webbing offers an excellent resource for genetic studies of spiders across families. Applications of the technique include species identification and monitoring, faunistic surveys, population connectivity, subpopulation structuring, and ex situ breeding programs.

Abstract Many spider species produce webs that represent a potential non-invasive source of DNA for conservation genetic analysis. Reported here is the successful isolation of target DNA from members of two families (Theraphosidae and Pholcidae) using a standard CTAB phenolchloroform-isoamyl protocol. The isolated DNA was of sufficient quality to permit routine PCR amplification and sequencing of mtDNA COI fragments of various sizes (maximum 710 bp attempted). This adds to other studies in demonstrating that webbing offers an excellent resource for genetic studies of spiders across families. Applications of the technique include species identification and monitoring, faunistic surveys, population connectivity, subpopulation structuring, and ex situ breeding programs.
Keywords Spider webs Á Ecological genetics Á Nondestructive sampling Á Non-invasive sampling Á Conservation Á Psalmopoeus Á Pholcus As DNA extraction techniques have improved, researchers in Arthropod conservation genetics have moved away from 'non-lethal sampling' (sampling of tissue which may impact the individual's future life but does not kill (Vila et al. 2009)) and begun to explore 'non-invasive sampling' (sampling which confers minimal costs to the individual but that is targeted to a specific species (Feinstein 2004)) and environmental DNA (eDNA, genetic material from bulk environmental samples, not necessarily targeted toward a taxonomic group (Barnes and Turner 2016)). Spider webbing represents a potential source of DNA for such applications (Xu et al. 2015).
DNA isolation was tested on samples of webbing from two species (Psalmopoeus cambridgei Pocock 1895, Theraphosidae, and Pholcus phalangioides Fuesslin 1775, Pholcidae) that produce different web forms. Psalmopoeus construct vertical sheet webs in enclosed spaces in trees, which are then covered in loose material surrounding the web structure; primarily detritus and leaves (Bushell pers. obvs.). Pholcus build 'space webs' which are used as preydetection structures from which the spider hunts prey (Jackson and Brassington 1987). Both species produce different web forms to species investigated previously (Latrodectus spp., Theridiidae, Xu et al. 2015).
Samples of captive Psalmopoeus cambridgei webbing were cleaned of large particles of detritus, but the majority of the fine detritus (pieces of prey, faeces from the spider, and local substrates etc.) remained stuck to the web, a potential source of non-target DNA (Xu et al. 2015). Webbing was cut into pieces to give individual sample weights of 2.3-8.5 mg.
Webs known to belong to Pholcus phalangioides were collected from a house in Wales (52.4113, -3.9897). Four samples of webbing without visible exoskeletons from either the web holder or prey were used, weighing 2.4-7.4 mg.
DNA from web samples was extracted using a standard CTAB phenol:chloroform:isoamyl alcohol (PCIA) method (Winnepenninckx et al. 1993), chosen for its relative low per-sample cost and applicability to low weight eDNA samples (Blake et al. 2015). Samples were placed in 350 ll of CTAB and 10 ll of Proteinase K and incubated overnight at 37°C with occasional vortexing. 350 ll of PCIA was then added and the mixture shaken for 20 min.
Following centrifugation at 15,000 RPM for 20 min, the upper aqueous phase was removed and subjected to an ethanol precipitation with 1 ml of absolute ethanol. Following a second precipitation using 1 ml of 70 % ethanol, eluted pellets were air dried and resuspended in 100 ll of ddH 2 O. Agarose gel electrophoresis of neat DNA solution revealed high molecular weight DNA in all cases. The concentration of the extracted DNA, estimated using a Nanodrop 2000 (ThermoScientific), was 15.6-23.3 ng/lL for the Psalmopoeus webbing, and 1.1-7.4 ng/lL for the Pholcus webbing, though these weights likely include DNA from prey and detritus (Xu et al. 2015).
Most PCRs produced amplicons of the expected sizes, with the largest amplicon being generated by the Folmer primers (710 bp) (Fig. 1), though this reaction failed for the Psalmopheus material. Only the smallest Pholcus phalangioides sample failed consistently to produce amplicons. Sequencing of amplicons was performed using AB BigDye technology. BLASTn confirmed species identify for both Psalmopoeus cambridgei and Pholcus phalangioides, demonstrating amplification of the target region and species.
This work demonstrates that large fragments of COI (710 bp) can be amplified from a range of spider webs, joining Xu et al. (2015) and Sint et al. (2015) in the recent push toward advancing Araneae conservation genetics. However, caution should be used when using universal primers for species surveys due to the potential mixture of species contained within the DNA extraction, ideally taxaspecific primers should be developed and used where possible. The large fragment of mtDNA suggests that this material will also permit genotyping of a range of nuclear markers such as microsatellites, AFLP etc., as shown by other studies on low-quality environmental DNA (Nyström et al. 2012;Calvignac-Spencer et al. 2013;Blake et al. 2015;Thomsen and Willerslev 2015). Studies on a variety of web producing species should greatly benefit from this technique, including work on DNA barcoding, ecological genetic surveys, and fine-resolution population connectivity. The number in the 'Psal' primer names denotes the position from the 5 0 end of a Psalmopoeus cambridgei CO1 sequence from GenBank (accession number JQ412455.1), whilst in the 'Phol' primers this denotes the position from the 5 0 end of a complete mitochondrial genome of Pholcus phalangioides from GenBank (accession number JQ407804.1). 'F' and 'R' at the end of the primer name refer to whether the primer is a forward or reverse respectively Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.