A new primer for metabarcoding of spider gut contents

Abstract

As a key predator group, spiders have received a lot of attention by food web ecologists. The difficulty involved in studying their diet has led to the use of new technologies such as metabarcoding of gut contents. The amplification of a broad range of spider prey without amplifying spiders themselves is challenging. Until now, an efficient universal primer for this purpose was not available. We developed a novel forward primer (NoSpi2) targeting the COI gene. The primer was designed not to amplify spiders of Pardosa genus while amplifying most other invertebrates. NoSpi2 was tested together with the reverse primer BR2 in silico, in vitro on single specimens of prey and spiders, on mock and malaise trap communities, and in an ecological application. In silico evaluation predicted high primer bias for Pardosa species and more generally for spiders of the oval calamistrum clade (Lycosidae and closely related species) and low bias for other invertebrates. These results were confirmed by in vitro tests. Additionally, some spider families were not amplified contrary to our expectations. We demonstrated a high efficiency for the primer pair NoSpi2/BR2 which recovered 94% of taxa in the mock community and 85% of the taxa detected by the best invertebrate primer pair known for the malaise trap community. The field experiment showed that Lycosidae (Hygrolycosa, Pardosa, Piratula, Trochosa) DNA is not amplified by NoSpi2/BR2. It demonstrated a broad range of detectable prey species (12 orders, 67 families, 117 species). The ability of NoSpi2/BR2 primer to reliably amplify prey species, without amplifying any predator DNA, makes it an ideal choice for gut content analysis for lycosid species and related species, even enabling the homogenization of entire specimens without dissection. Given that the detected prey species included other spiders and carabid beetles, this primer could be also used to study intraguild predation.

Deciphering the diet of a wandering spider (Phoneutria boliviensis; Araneae: Ctenidae) by DNA metabarcoding of gut contents

“…The development of the blocking primers was carried out following the protocol established by Lafage et al (2020). Mitochondrial cytochrome c oxidase subunit 1 (COI) sequences for 230 entries of Ctenidae spiders were downloaded from BOLD database (https:// www.bolds ystems.org/) and clustered using the "PrimerMiner" package v0.18 (Elbrecht & Leese, 2017).…”

“…PCRs were carried out in 25 µl reaction volumes containing 2 µl of DNA extract with equal DNA concentrations (39 ng/µl), 12.5 µl of MyTaq mastermix (Biolone), and 2.5 µM of each primer (dgHCO and mlCOIntF primers (Leray et al, 2013) to amplify the COI region. Four different annealing temperatures were tested (40, 40.3, 40.9, and 48°C) in the preliminary assays which were previously shown to work well for COI (Lafage et al, 2020). The optimum temperature being determined as 48°C (Figure S1).…”

“…Consequently, digestion takes place in many different parts of the body and dissecting the whole gut is challenging due to its complexity and extent (Macías-Hernández et al, 2018). This condition thus restricts studies of spider diet (Birkhofer et al, 2017;Jackson et al, 2001;Lafage et al, 2020;Pompanon et al, 2012). Diet studies involving the taxonomic determination of prey items by field observation depend largely on the experience of the researcher, and are therefore not only labor-intensive, but potentially biased.…”

“…Molecular gut content analysis is a valuable tool for characterizing trophic interactions where conventional diet records are difficult to establish. For the analysis of spider diet, DNA-based methods thus possess a range of advantages over the classical approaches in diet analyses (Lafage et al, 2020;Sheppard & Harwood, 2005).…”

“…DNA metabarcoding has proven to be a very accurate and efficient tool for the analysis of spider diets, capable of complementing other techniques for the detection of prey, and detecting previously unnoticed prey in ecological studies on multiple species of spiders with euryphagous diets (Lafage et al, 2020;Piñol et al, 2019).…”