Sample collection
Copepods were collected on four seasonal cruises from the central
Barents Sea to the Arctic Nansen basin north-east of Svalbard, Norway
(Table S2). Cruises occurred during Autumn (5 – 27 August 2019), early
winter (28 November – 17 December 2019), late winter (2 – 24 March
2021) and early spring (27 April – 20 May
2021), and visited three stations
on the Barents Sea central shelf (76.00N, 31.22E), northern shelf
(79.72N, 34.32E) and Nansen basin (81.83-82.16N, 28.15-29.84E, positions
varied due to sea-ice drift, Fig. S1). Small-sized mesozooplankton
(<1 mm) were collected in vertical 64 µm Bongo-net hauls (to
full depth or max. 1000 m, ascent = 0.3 m s-1, descent
= 0.5 m s-1, 60 cm mouth diameter). All large and/or
gelatinous animals (1-10 cm) were removed, and the remaining suspension
sieved (64 µm) to discard seawater. Ice-cold ethanol (96%, -20 °C) was
then used to rinse retained mesozooplankton, before transfer into a
sample bottle. The container was topped up with ice-cold ethanol and
stored at -20°C.
Microsetella norvegica(Boeck, 1865), Microcalanus spp. (M. pygmaeus or M.
pusillus , Sars G. O., 1900-1903) and Oithona similis (Claus,
1866) were morphologically identified under a stereomicroscope (Table
S2). Up to 14 individuals per species and station were picked where
available. Each specimen was thoroughly rinsed individually three times
in Milli-Q water, transferred to tissue lysis buffer (E.Z.N.A Tissue DNA
kit, Omega Bio-tek). Surface sterilization with bleaching was excluded,
since the minute body size (< 1 mm) of the copepods analyzed
herein raised concerns regarding how the treatment could penetrate and
potentially alter the dietary signal. Also, existing literature, mostly
based on arthropods, are in disagreement regarding the efficacy of
bleaching, with one study indicating little effect on the overall
dietary signal (Miller-ter Kuile et al., 2021). DNA extraction was
performed per manufacturer’s protocol (“Tissue Spin Protocol”,
E.Z.N.A® Tissue DNA kit, Omega Bio-Tek), with a
lowered elution volume of 2 x 50 µL elution buffer. One negative without
material was included with every round of extraction.
We checked all copepod and
negative DNA extracts by PCR amplification of a 18S V7 fragment
(~240 bp) using the universal eukaryotic primers 960F
(5’-GGCTYAATTTGACTCAACRCG-3’) and modified 1200R
(5’-GGGCATCACAGACCTG-3’) (Cleary & Durbin, 2016; Gast et al., 2004,
Table S1). The amplifications were
performed in 10 µL reaction volumes (4.8 µL MQ water, 1.0 µL DreamTaq
buffer (10X), 0.2 µL DreamTaq Polymerase (5 U µL-1),
1.0 µL dNTP (2.5 mM each), 0.5 µL forward-primer (960F, 10 µM), 0.5 µL
reverse-primer (1200R, 10 µM) and 2.0 µL template). PCR negatives had 2
µL MQ instead of template. Thermal cycling consisted of an initial
denaturation (2 min, 95°C), and 35 cycles of denaturation (30 s, 94°C),
annealing (30 s, 54°C), and elongation (30 s, 72°C), with a final
elongation step (15 min, 72°C). The success of the amplifications was
inspected by 1% agarose gel electrophoresis, confirming single band PCR
products in all samples and no amplification in all extraction
negatives.