Gut content of Antarctic krill Euphausia superba from the West Antarctic Peninsula

Study Extent

Euphausia superba specimen were collected during the cruise NBP1410 of the US RV Ice Breaker ‘Nathaniel B. Palmer’ between 10 and 21 December 2014.

Sampling

Krill were collected from the water column with oblique tows of a 1meter squared multiple opening closing net and environmental sensing system (MOCNESS). Net mesh size was 333 μm. Water samples were collected at 10 m depth for DNA analysis of the community composi- tion. Duplicate subsamples of 100 ml of water from each of the sampled areas were filtered onto 0.2 μm pore size, 25 mm diameter membrane filters under gentle vacuum pressure.

Quality Control

Agarose gels with ethidium bromide were used to confirm all sample reactions had produced amplicons of the expected size, and that no amplification was visible in any of the negative controls.

Method steps

1. Krill were processed immediately and preserved in 95% reagent grade ethanol. Water filters for DNA analysis were pre- served at −80°C, and were maintained frozen until DNA extraction.
2. DNA was extracted with the DNeasy blood and tissue kit (Qiagen) as per manufacturer’s directions. Krill stomachs were first broken open with a sterilized toothpick to ensure the contents would fully lyse. For water filters, double volumes were used of lysis buffers to ensure the filter was submerged and thus fully lysed. DNA from krill and water filters were never extracted on the same days.
3. A nested PCR approach was used to amplify the v7 region from all non-krill 18S rDNA. In the first PCR, krill stomach DNA extracts were amplified with universal 18S primers, while krill’s own DNA was blocked with a krill-specific PNA probe. These reactions included 1× GoTaq Green Master Mix (Promega), 0.5 μM each forward and reverse primers (forward: 5’-GGG CAT CAC AGA CCT G-3’, reverse: 5’-GGC TYA ATT TGA CTC AAC RCG-3’; modified from Gast et al. 2004 as per Cleary et al. 2016), 20 μM krill-specific PNA (from 100 μM stock re-suspended in 2.5% trifluoroacetic acid, 5’-CGT CGG GTT GTC TTG-3’; Cleary et al. 2012), and 20% by volume stomach DNA extract. DNA was used at extracted concentrations. Thermocycling consisted of 95°C for 30 s, followed by 30 cycles of 94°C for 30 s, 67°C for 30 s, 58°C for 30 s, 60°C for 30 s, with a final extension at 60°C for 5 min and then immediate cooling of the reactions to 4°C. In the second PCR, the stomach contents amplicons were re-amplified for only a few cycles with the primers containing the illumina adaptors, in order to add these adaptor sequences onto the ends of the amplicons. These reactions contained 1× GoTaq Green Master Mix, 0.2 μM each forward and reverse primers and 20% by volume amplicons from the first round of PCR
4. Illumina primers were reversed, which is to say the Read1 adaptor (traditionally forward) was put on the reverse primer, and the Read2 adaptor on the forward primer. Thermocycling for these reactions consisted of 94°C for 30 s, followed by 10 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, with a final extension of 72°C for 5 min and then immediate cooling to 4°C.
5. A final PCR of 5 cycles was performed by the sequencing center to attach further sequencing adaptors and individual tags, and samples were combined into 2 sequencing pools. Krill individuals from each of the sampling locations were divided between the 2 pools; all captive krill were placed in the same pool. Each pool was sequenced in one lane on 2 separate Illumina MiSeq runs, with V2 chemistry for 200 bp in each direction.

Antarctic Peninsula: Adelaide Island, Andvord Bay, Anvers Island, Flandres Bay, Gerlache Strait, Lemaire Strait and Palmer Deep