Molecular gut content analysis of different spider body parts

Abstract

Molecular gut-content analysis has revolutionized the study of food webs and feeding interactions, allowing the detection of prey DNA within the gut of many organisms. However, successful prey detection is a challenging procedure in which many factors affect every step, starting from the DNA extraction process. Spiders are liquid feeders with branched gut diverticula extending into their legs and throughout the prosoma, thus digestion takes places in different parts of the body and simple gut dissection is not possible. In this study, we investigated differences in prey detectability in DNA extracts from different parts of the spider´s body: legs, prosoma and opisthosoma, using prey-specific PCR and metabarcoding approaches. We performed feeding trials with the woodlouse hunter spider Dysdera verneaui Simon, 1883 (Dysderidae) to estimate the time at which prey DNA is detectable within the predator after feeding. Although we found that all parts of the spider body are suitable for gut-content analysis when using prey-specific PCR approach, results based on metabarcoding suggested the opisthosoma is optimal for detection of predation in spiders because it contained the highest concentration of prey DNA for longer post feeding periods. Other spiders may show different results compared to D. verneaui, but given similarities in the physiology and digestion in different families, it is reasonable to assume this to be common across species and this approach having broad utility across spiders.

High-throughput sequencing for community analysis: the promise of DNA barcoding to uncover diversity, relatedness, abundances and interactions in spider communities

“…This approach accommodates thousands of samples in a single sequencing run. Multiplex PCRs targeting multiple unlinked loci can additionally reduce the necessary number of PCRs (Krehenwinkel et al 2018;Macías-Hernández et al 2018), and inline barcodes attached to the first-round PCR primers allow for a further increase in sample number (Sternes et al 2017). Alternatively, fusion primers including sample tags and sequencing adapters can be used.…”

“…However, this method will only work well if the predator DNA is not degraded; therefore, it is not suitable for old or poorly preserved specimens. An additional enrichment of prey DNA can be achieved by extracting DNA from the spider's opisthosoma only, which contains the majority of the animal's digestive tract (Krehenwinkel et al 2017b;Macías-Hernández et al 2018). Fig.…”

Bats as potential suppressors of multiple agricultural pests: A case study from Madagascar

“…The reverse primer was jgHCO2198 (5'-TAIACYTCIGGRTGICCRAARAAYCA-3'; (Geller et al, 2013)). The Leray fragment has already been successfully applied to the characterisation of both marine fish gut contents (Leray et al, 2013), marine invertebrates (Siegenthaler et al, 2018) and terrestrial arthropods (Macías-Hernández et al, 2018). A single-PCR step using primers with attached eight-base oligo-tags (Coissac et al, 2012) was used to label different samples in a multiplexed library; moreover a variable number (2, 3 or 4) of fully degenerate positions (Ns) was added at the beginning of each primer, in order to increase variability of the amplicon sequences (Guardiola et al, 2015) .The PCR mix recipe included 10 μl AmpliTaq gold 360 master mix (Applied Biosystems), 3.2 μg Bovine Serum Albumin (Thermo-Scientific), 1 μl of each of the 5 μM forward and reverse tagged-primers, 5.84 μl water and 2 μl extracted DNA template (~ 5 ng μl-1).…”

Advancing the integration of multi‐marker metabarcoding data in dietary analysis of trophic generalists

“…Furthermore, metabarcoding makes it possible to identify virtually all species consumed by a predator or herbivore, including rare food items (Hope et al, 2014;Nielsen et al, 2017;Razgour et al, 2011;Soininen et al, 2009), though this is conditional on DNA quality and the availability of DNA reference databases (Deagle, Eveson, & Jarman, 2006;Elbrecht et al, 2016;Gerwing, Kim, Hamilton, Barbeau, & Addison, 2016). Due to its strengths and cost-effectiveness, this approach has been increasingly used to describe the diet of many animals (Deagle et al, 2019;Kaunisto, Roslin, Sääksjärvi, & Vesterinen, 2017;Macías-Hernández et al, 2018;Soininen et al, 2009) and even carnivorous plants (Littlefair, Zander, Sena Costa, & Clare, 2019). However, there are still significant uncertainties regarding potential biases and pitfalls of metabarcoding, and how best to address them, which may significantly impact on the results of dietary analysis (Nielsen et al, 2017).…”