A new primer for metabarcoding of spider gut contents
- Published
- Accepted
- Subject Areas
- Agricultural Science, Ecology, Ecosystem Science, Entomology, Molecular Biology
- Keywords
- Zoropsidae, Ctenidae, Pisauridae, Oxyopidae, Senoculidae, gut content metabarcoding, Lycosidae, molecular diet analysis, Psechiridae, Trechaleidae
- Copyright
- © 2019 Lafage et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2019. A new primer for metabarcoding of spider gut contents. PeerJ Preprints 7:e27854v1 https://doi.org/10.7287/peerj.preprints.27854v1
Abstract
As a key predator group, spiders have received a lot of attention by food web ecologists in diverse fields such as pest control, pollutant transfers, and cross-ecosystem fluxes. 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 and, until now, an efficient universal primer purposed for this has not existed.
We developed a novel forward primer (NoSpi2) targeting the cytochrome c oxidase subunit I gene. The primer was designed not to amplify spiders of the oval calamistrum clade (Lycosidae and closely related species) while still 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 spiders of the oval calamistrum clade and low bias for all other invertebrates. These results were largely 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 up to 94% of taxa in the mock community and 85% of the taxa detected by the best invertebrate primer pair known (BF3+BR2) for the malaise trap community. The field experiment showed that Lycosidae spider DNA is not amplified by the NoSpi2 primer set. It also demonstrated a broad range of detectable prey species. We found prey from 12 orders, 67 families and 117 species.
The ability of the NoSpi2/BR2 primer combination to reliably amplify prey species, without amplifying any predator reads, makes it an ideal choice for gut-content analysis for spider species of lycosids and closely related species, even enabling the homogenization of entire spider specimens without dissection. Given that the detected prey species included other spiders and carabid beetles, this primer could be used for not only diet and biological control studies, but also to study intra-guild predation.
Author Comment
This is the first version of the paper. It will be submitted in the days to come to Methods in Ecology and Evolution
Supplemental Information
Picture of the gel electrophoresis obtained after PCR with NoSpi2/BR2
Coll: Collembola (Poduridae), Trico1: Trichoptera (Limnephiliidae), Trico2: Trichoptera (Phryganeidae), Trico3: Trichoptera (Polycentropodidae), Pleco: Plecoptera (Nemouridae), Carab1: Carabidae (Bembidion bruxellens), Neg: negative control
Picture of the gel electrophoresis obtained after PCR with NoSpi2/BR2
Eph1: Ephemeroptera (Baetidae), Eph2: Ephemeroptera (Leptoplebiidae), Eph3: Ephemeroptera (Leptoplebiidae), Megalo: Megaloptera, Carab2: Carabidae (Bembidion sp.), Carab3: Carabidae (Elaphrus riparius), Pardo abdo: starved Pardosa abdomen
Picture of the gel electrophoresis obtained after PCR with NoSpi2/BR2
Dipt1: Dipatera (Chironomidae), Dipt2: Diptera (Chaboridae), Dipt3: Diptera (Simulidae), Dipt4: Diptera (Simulidae), Pardo1: Lycosidae (Pardosa amentata), Pardo2: Lycosidae (Pardosa lugubris), Pardo+Coll: starved Pardosa + collembola, Neg: Negative control
Picture of the gel electrophoresis obtained after PCR with NoSpi2/BR2
Picture of the gel electrophoresis obtained after PCR with NoSpi2/BR2
Pardo3: Lycosidae (Pardosa prativaga), Pardo4: Lycosidae (Pardosa pullata), Pirat: Lycosidae (Piratula hygophila), Tetra: Tetragnathidae (Tetragnatha sp.), Liny1: Linyphiidae (Tenuiphantes cristatus), Liny2: Linyphiidae (Bathyphantes nigrinus), Pardo+Tricho: starved Pardosa + Trichoptera, Pardo + Pleco: starved Pardosa + Plecoptera
Picture of the gel electrophoresis obtained after PCR with NoSpi2/BR2
1: Thomisidae (Diaea dorsata), 2: Theridiidae (Enoplognatha ovata), 3: Araneidae (Araneus alsine), 4: Pisauridae (Pisaura mirabilis), 5: Hahniidae (Hahnia nava), 6: Cybaeidae (Cryphoeca sylvicola), 7: Tetragnathidae (Metellina merianae) , 8: Salticidae (Neon reticulatus) , 9: Oxyopidae (Oxyopes salticus), 10: Ctenidae, Neg: negative control
Summary table of all the species detected in spider gut contents during the field test
# detect: number of spiders in which a prey was detected. # reads: total number of reads in the library. Sequences were attributed at species level with a similarity match ≥ 98%, to the genus level with a similarity match ≥ 95%, to the family level with a similarity match ≥ 90% and to the order level with a similarity match ≥ 85%.