Genetic analysis of eel gut contents, marine snow aggregates, and plankton specimens

The 16S and 18S rRNA marker genes were examined with Illumina MiSeq to discern prokaryotic and eukaryotic compositions, respectively, of eel guts and marine snow aggregates, while Sanger sequencing of 18S rRNA genes from zooplankton specimens was used to generate the reference database. Total genomic DNA was extracted from each eel gut using the E.Z.N.A. Tissue DNA Kit (OMEGA Bio-Tek, Georgia, USA), and from the marine snow aggregates using phenol-chloroform extraction⁵⁹, and quantified (PicoGreen®, Quant-iT^TM, Invitrogen, ThermoFisher Scientific, Massachusetts, USA). The V7 region of the 18 S rRNA genes was PCR amplified using 0.05 units µl⁻¹ MyTaq DNA polymerase (Saveen & Verner AB, Limhamn, Sweden), ca. 0.08 ng µl⁻¹ DNA template, and 0.5 µM of each of universal primers UnivF-1183mod and UnivR-1443mod⁶⁰ indexed for Illumina sequencing in 25 µl reaction volumes. A specially designed blocking primer, modified with a C3-spacer to prevent elongation (cf.⁶¹), was introduced to each PCR mixture with an eel gut DNA template to block the amplification of A. anguilla DNA (5′-CATCACAGACCTGTTATTGCTCAATCTCGTGTGGCTGAACG3–3′; 3 = C3-spacer), while still allowing amplification of the full range of potential prey organisms identified in Riemann et al.²⁵. Sequences from a range of potential prey organisms found by Riemann et al.²⁵ were aligned and analyzed as done by Leray et al.⁶² to facilitate blocking primer design. The blocking primer was added at a ratio of 0.8:1 relative to the reverse primer, after trials to determine a sufficient host-blocking effect utilizing the least amount of blocking primer. Blocking primer trials were conducted against DNA of genetically-confirmed A. anguilla specimens from a previous study⁶³. PCR conditions were 95 °C for 2 min followed by 30 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 45 s, and termination at 72 °C for 10 min. Triplicate PCR reactions per sample were pooled, purified (Agencourt AMPure XP magnetic bead system, Beckman Coulter Life Sciences, Indiana, USA), and quantified using PicoGreen. Finally, PCR amplicons from eel guts and marine snow aggregates were pooled at equimolar concentrations, and submitted for paired-end sequencing on an Illumina MiSeq V2 2 × 250 (University of Copenhagen, Denmark).

The V3 and V4 regions of the 16S rRNA genes were PCR amplified with the universal primers 341F and 806R⁶⁴. Reactions were set up in 20 µl using 0.03 units µl⁻¹ Phusion HotStart II polymerase and HF buffer (Thermo Fisher, Massachusetts, USA), ca. 0.1 ng µl⁻¹ DNA template, and 100 ng µl⁻¹ bovine serum albumin (Bioron, Ludwigshafen, Germany). PCR conditions were 98 °C for 30 s followed by 35 cycles of 98 °C for 10 s, 56 °C for 20 s, and 72 °C for 30 s, and final elongation at 72 °C for 5 min. Sequencing and indexing adaptors were added to the PCR products by a second round of PCR, performed by applying the Nextera indexing kit (Illumina Inc. San Diego, California, USA) under the conditions described earlier⁶⁵, but modified to 5 μL of the 1st round PCR product as the template and 17 cycles. PCR products were purified with MagBio magnetic beads (MAGBIO GENOMICS, Maryland, USA). PicoGreen quantification was used for molarity estimation and samples were paired-end sequenced on an Illumina MiSeq platform using the MiSeqV2 500 cycles sequencing kit (Aarhus University, Roskilde, Denmark).

DNA for the reference database was extracted (E.Z.N.A. Tissue DNA Kit) from individual planktonic organisms, quantified (PicoGreen), and PCR amplified using universal 18 S rRNA primers F-1183mod and R-1631a according to Hadziavdic et al.⁶⁰. To increase the reference-database coverage, DNA from 14 genetically-confirmed fish specimens from 13 families, including 3 common eel families, was amplified with the same primers. These fish represent the most abundant fish species known to inhabit this area of the Sargasso Sea at the time of sampling⁶⁶. PCR amplicons were purified (E.Z.N.A. Cycle Pure Kit, OMEGA Bio-Tek, Georgia, USA) and Sanger sequenced commercially (Eurofins, Ebersberg, Germany).