DNA Extraction and amplification

Genomic DNA was extracted using the JetQuick DNA Kit (GENOMED), following the protocol for purification of total DNA from animal tissues, with some adaptations. The first change to the standard protocol was the volume of reagents used to digest the recovered content: a total of 4 ml of lysis buffer was added to 2 ml of gut content of each individual, along with 60 μl of proteinase K, in a 15 ml tube; the samples were incubated overnight at 56 °C. Due to the initial large volume, several repetitions of the following steps of the protocol had to be performed. In addition, DNA from sunfish muscle and that of the pelagic crab, Polybius henslowii, which was frequently observed in the gut contents, was also extracted, following the standard protocol, and were used as controls for the subsequent polymerase chain reactions (PCR). Extracted DNA was immediately frozen at −20 °C until PCR was carried out.

Despite being controversial, the universal COI (cytochrome oxidase subunit I) primers are still recommended as a metabarcoding marker, particularly when species-level identification is critical, as in our study⁵³. Hence, we used degenerate versions of the COI primers LCO1490 and HCO2198⁵⁴, the jgLCO1490 (5′ TITCIACIAAYCAYAARGAYATTGG 3′) and the jgHCO2198 (5′ TAIACYTCIGGRTGICCRAARAAYCA 3′)⁵⁵. To quantify the amplification of sunfish DNA, PCR was performed for a random subset of 10 individuals and sequencing through cloning (10 clones per individual). Since over 60% of the sequences retrieved belonged to the sunfish, we opted to design a blocking primer 5′-CAAAGAATCAGAAGAGATGTTGA [SpcC3]-3′ based on the mitochondrial genome of sunfish available on GenBank (Accession number {"type":"entrez-nucleotide","attrs":{"text":"AP006238","term_id":"46092354","term_text":"AP006238"}}AP006238). This primer overlapped with the 3′ end of the reverse universal primer, but extended into the sunfish specific sequence, and was modified with a C spacer following 33, to prevent elongation without affecting the annealing properties. Additionally, we split the samples into four different fish size classes (<0.40 m; 0.40–0.60 m; 0.60–0.80 m and >0.80 m TL), to better characterise the sunfish diet with growth and to detect possible changes in the dietary habits. Thus, in PCRs to be sequenced through NGS we used four different multiplex identifiers from Roche (MIDs) attached to the original primer sequences (MID001: ACGAGTGCGT; MID002: ACGCTCGACA; MID003: AGACGCACTC; MID004: AGCACTGTAG).

For DNA amplification, 1 μl of the extracted DNA was added to a total of 19 μl of PCR reaction mix and proof reading Platinum Taq (Invitrogen) was employed in all PCR reactions (see Supplementary Table S2 for PCR mix descriptions). Firstly, we tested the efficiency of two different concentrations of blocking ^primer [10 and 20x] relative to the universal COI primers, to prevent sunfish DNA amplification during PCR. We found that a proportion of 20:1 of blocking primer was needed to ensure minimum sunfish DNA amplification.

A touchdown PCR amplification protocol was implemented due to the higher temperature demand of the blocking primer to bind to the sunfish DNA. Thermal cycling conditions were as follows: initial denaturation at 95 °C for 5 minutes followed by 62 cycles of: 95 °C for 30 seconds; annealing temperature step-downs every 4 cycles of 1 °C (from 55 °C to 47 °C) until the temperature reached 46°C which was maintained for 20 cycles; 72 °C for 30 seconds; and a final extension at 72 °C for 7 minutes. DNA amplification of the contents and P. henslowii, and absence of amplification for the sunfish DNA and PCR reagents negative control (blank), were verified in an agarose gel (e.g. Supplementary Fig. S1). PCR reactions used in NGS were repeated at least three times per sample, and were pooled as explained in the next section.