Amplification success was verified by running 2 ОјL of PCR product on a 2% agarose gel. Thermocycling conditions included an initial denaturing step at 95 вЊ for 15 min, followed by 40 cycles of denaturing at 95 вЊ for 30 s, annealing at 45 вЊ for 60 s, extension at 72 вЊ for 30 s, and a final extension at 72 вЊ for 10 min. Initial PCR mixes comprised 10 ОјL of Qiagen Multiplex PCR Master Mix, 0.3 ОјL or 0.4 ОјL of 10 mM target primer (fwh1 and LerayXT, respectively), 8.0 ОјL of 10 mM blocking primer, 2 ОјL of DNA template, and enough water for a final volume of 25 ОјL. Salamandra salamandra COI sequences from NCBI (KX094979 & GQ380404) were used to design blocking primers for fwh1 (Ssal_fwhF1-blk 5вЂІ-CAA AGA CAT TGG CAC CCT CTA CCT AAT TTT TGG -3вЂІ) and LerayXT (Ssal_mlCO1intF-blk 5вЂІ-GAA CAG TCT ACC CCC CCC TTG CCG GAA ATC TGG -3вЂІ). To mitigate primer bias, two COI-specific primer pairs-fwh1 (fwhF1 5вЂІ-YTC HAC WAA YCA YAA RGA YAT YGG-3, fwhR1 5вЂІ-ART CAR TTW CCR AAH CCH CC-3вЂІ) and LerayXT (jgHCO2198 5вЂІ-TAI ACY TCI GGR TGI CCR AAR AAY CA-3вЂІ, 185 bp mlCOIintF-XT 5вЂІ-GGW ACW RGW TGR ACW ITI TAY CCY CC-3вЂІ 313 bp) -were used. While 18S has proven useful to detect potential prey items, the use of more informative (i.e., variable) DNA fragments such as the cytochrome c oxidase I (COI) benefits from a large reference database that is supported by the Barcode of Life Data System (BOLD), and the relatively high variability of the region allows for high-resolution taxonomic assignment. used the 18S ribosomal RNA (18S) region to characterize the diets of adult fire salamanders ( Salamandra salamandra) (Linnaeus, 1758) by collecting fecal samples from three Belgian forests. To our knowledge, only one study has applied DNA metabarcoding to investigate the diet of adult salamander species. Similar to visual inspections, DNA metabarcoding can also indicate the preferential diets of salamanders and their role as predators in communities, and can inform us about invertebrate biodiversity. This COI metabarcoding study provides recommendations and resources for subsequent research using DNA metabarcoding to study amphibian diets.ДNA metabarcoding can help to identify prey species that are consumed over a longer period of time, avoids the detection bias against soft-bodied prey, and requires less taxonomic training in prey identification. We observed differences in the prevalence of prey identified between sampling regions both in our study and in other studies of S.

Between primers, there were no significant differences in the diversity and richness of prey detected. Our COI metabarcoding approach increased taxonomic resolution and supported a generalist diet in S. Two COI primers were used in combination to investigate the potential impact of primer bias in prey detection. To test the efficiency of COI-based metabarcoding in determining salamandersвЂ™ diet diversity, we compared our COI-based results with results from traditional diet studies from neighboring and distant populations, as well as with recent findings obtained in a DNA metabarcoding study using 18S.

Here, we used DNA metabarcoding to characterize the diet of fire salamanders ( Salamandra salamandra) (Linnaeus, 1758) in three distinct regions across the northwestern Iberian Peninsula. Although DNA metabarcoding provides numerous benefits in characterizing diet-such as detecting prey animals that are difficult to visually identify-this method has seen limited application in amphibian species. DNA metabarcoding has proven to be an accessible, cost-effective, and non-invasive tool for dietary analysis of predators in situ.

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