MEDITERATRI Project (HRZZ) - Trophic interactions of spiders in Mediterranean agriculture using DNA metabarcoding
We hypothesized that spiders, as abundant predatory arthropods in Mediterranean agriculture, can provide biocontrol services in olive groves and vineyards. For that, their diets and prey choices in agricultural sites, with integrated (IPM) and ecological pest management (EPM) within Zadar County, Croatia. Spider were sampled during spring and autumn in 2018 and 2019, by hand or using live pitfall traps. A total of 256 spiders were collected and included in eDNA of gut contents analysis. The DNA was extracted from the abdomen of each individual. Two pairs of primers were selected for dietary metabarcoding of spider guts: the primer pair BF3-BR2 that amplifies the 418 bp-long COI target region and the TelperionF-LaurelinR that amplifies the 301 bp COI region. Illumina library was prepared at Novogene Europe (Cambridge, UK) by including Illumina Nextera adapters. Next-generation sequencing was performed on an Illumina Novaseq high-throughput instrument, with an amplicon length of 250 bp in both directions with a sequencing depth of 35,000 reads per sample. Sequencing on Illumina generated 9,234,272 reads for sequencing with the BF3-BR2 primer pair and 3,519,228 reads for sequencing with the TelperionF-LaurelinR primer pair. In FastP all reads underwent quality checks and trimming. Sequences were denoised and grouped into operational taxonomic units (OTUs) using Unoise3 in Usearch11. A taxonomic identity was done using data from GenBank after being proceeded with BLASTn v2.7.1 and a 97% identity threshold. Results showed strong dietary differences in spider guilds (; ground hunters, ambush hunters, other hunters and web weavers). Thee prey detected in their gut that made up 77% of the overall diet of spiders belong to insect orders: Diptera, Lepidoptera, Hemiptera, and Hymenoptera (mainly ants). We demonstrate that management type, as well as guild type, significantly influenced predation on phytophagous insects. More individuals consumed phytophagous insects in sites with an ecological type of management suggesting a higher biocontrol potential in sites with EPM. On the other hand, the overall predation on phytophagous insects remained consistent across seasons, suggesting a combined constant rate of predation in both management types. This is the first study using a DNA metabarcoding approach in Croatia for analysing spiders' trophic interactions.
Steps to reproduce
The bioinformatic pipeline of Drake et all. (2022) were used in analysing the high throughput sequences derived from an Illumina Novaseq high-throughput instrument, with an amplicon length of 250 bp in both directions with a sequencing depth of 35,000 reads per sample. Two primer pars were used in PCR: BF3-BR2 and TelperionF-LaurelinR primer pair. In FastP , All reads were checked for quality checks and trimming using FastP (Chen et all 2018). The read pairs were demultiplexed using Mothur v1.39.5 (Schloss et all. 2009); the indices of 8bp, MID tags, and primer sequences were removed; the reads were demultiplexed to generate one file per sample ID. The sample ID was added to the read headers and reads were then compiled into a single file. Sequences were denoised and grouped into operational taxonomic units (OTUs) using Unoise3 in Usearch11 (Edgar 2010). Taxonomic identification were done using BLASTn v2.7.1 (Camacho et all. 2009) and data from the GenBank -NCBI (The National Center for Biotechnology Information), with 97% identity threshold. Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos, J.; Bealer, K.; Madden, T.L. BLAST+: Architecture and applications. BMC Bioinform. 2009, 10, 421. Chen, S.; Zhou, Y.; Chen, Y.; Gu, J. Fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018, 34, i884–i890. Drake, L.E.; Cuff, J.P.; Young, R.E.; Marchbank, A.; Chadwick, E.A.; Symondson, W.O. An assessment of minimum sequence copy thresholds for identifying and reducing the prevalence of artefacts in dietary metabarcoding data. Methods Ecol. Evol. 2022, 13, 694–710. Edgar, R.C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 2010, 26, 2460–2461. Schloss, P.D.; Westcott, S.L.; Ryabin, T.; Hall, J.R.; Hartmann, M.; Hollister, E.B.; Lesniewski, R.A.; Oakley, B.B.; Parks, D.H.; Robinson, C.J.; Sahl, J.W.; Stres, B.; Thallinger, G.G.; Van Horn, D.J.; Weber, C.F. Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 2009, 75, 7537–7541.
Hrvatska Zaklada za Znanost
MEDITERATRI Project (HRZZ UIP-2017-05-1046)
Unity through Knowledge Fund
2A Grant Agreement No 10/19 granted to Lucija Šerić Jelaska
Hrvatska Akademija znanosti i umjetnosti