Food webs - 2020

Sampling was conducted in Kematen in Tirol, Austria, where spring barley (Hordeum vulgare L.) was grown in six fields that have had organic management for the past ten years, three in 2020 and 2021, respectively. Each field was split in half, one side being fertilized with manure and the other remaining unfertilized, as a negative control. The manure treatment was independently applied by each field’s respective owner, at a standard rate of 1.5 metric tons (1 500kg) per hectare, using manure spreaders, after tilling and pressing the soil. Four 5x5m plots per treatment were delimited, to carry out all sampling. These plots had no barriers or fences surrounding them that would impede or limit movement between the inside of the plots and the rest of the field area. Additionally, the plots were at least 5m from the field edge, to avoid edge effects. They were at least 10m way from each other along the field’s width, and between 15 to 20m along the length of the field. To avoid any spillover of treatment effects, plots were set at least 25m away from the border between the two treatments within the same field.Sampling was conducted every two weeks, with one week of sampling and one of rest. The sampling period started on the 21st of April and ended on the 14th of July in 2020, while it started on the 3rd of May and ended on the 16th of July in 2021, to encompass almost the entire crop season. This resulted in seven sampling time points for the first year and six for the second.A total of 2404 ground beetles, 913 rove beetles and 567 spiders were captured in 2020, and 1977 ground beetles, 891 rove beetles and 250 spiders in 2021. The beetles’ gut content and the spiders’ full bodies were extracted with a BioSprint 96 DNA Blood Kit (Qiagen, Hilden, Germany) on a QIAGEN Biosprint96® workstation for automated DNA extraction, following the manufacturer’s recommendations. After extraction, three different multiplex-PCR assays were run per sample. The first assay targeted several herbivore and detritivore taxa (assay in Rennstam Rubbmark et al. 2019), the second targeted generalist and specialist predators, such as spiders and ladybeetles, as intra- and extraguild predation (primers from Sint et al. 2014 and Staudacher et al. 2016). Lastly the third assay identified the genus of beetles consumed, also as intraguild predation, from a selected set of common taxa consisting of Bembidion spp., Harpalus spp., Poecilus spp., Pterostichus spp., Philonthus carbonarius and Philonthus cognatus. The intraguild interactions, and implications on competition, have been analysed in greater detail elsewhere (Leote et al. 2024).<br>Following Rennstam Rubbmark et al. (2019) a diagnostic multiplex-PCR was chosen over metabarcoding, as our regurgitate samples contain not only different amounts of DNA from several food sources, but also a lot of consumer DNA. This drastically lowers the detection of food DNA, as the primers targeting consumed taxa also bind to the taxonomically closely related consumer. Therefore, to ensure a more reliable and robust consumed DNA detection, we employed the three PCR assays described above and in the supplementary tables.After the multiplex-PCRs, the samples were screened using capillary electrophoresis on an QIAxcel Advanced system and the ScreenGel software, with a DNA Screening Kit (2400) using a 15-3k base pair alignment marker, following the manufacturer’s recommendations. The screening profile on ScreenGel was set to the standard AM320 method, with the relative fluorescence units (RFU) detection threshold defined at 0.07, as opposed to the default 0.1, to account for the fact the samples were extracted from gut content and thus partially digested.