Krill Fatty Acid Data

Brief description

The fatty acid content and composition of the Antarctic krill Euphausia superba Dana, 1850 were investigated using samples collected by a commercial fishing vessel. This dataset allowed comparison between seasons, years (2013–2016), and different fishing locations. Quantities of omega 3 fatty acids 20:5n-3 and 22:6n-3 (mg/g dry mass; DM) were highest in autumn and decreased through winter to reach a spring low. Quantities of the flagellate marker 18:4n-3 and diatom marker 16:1n-7c were variable and did not display the same seasonal fluctuations. In summer, krill had high percentages (% total fatty acids) of 20:5n-3 and 22:6n-3, total PUFA, and low 18:1n-9c/18:1n-7c ratios, indicating a more herbivorous diet. Krill became more omnivorous from autumn to spring, indicated by increasing ratios of 18:1n-9c/18:1n-7c and percentages of Σ 20:1 + 22:1 isomers. Bacterial fatty acids (Σ C15 + C17 + C19 isomers) were minor components year-round (0.9–1.8 %). Seasonal levels of herbivory and omnivory differed between years, and levels of specific fatty acid ratios differed between fishing locations. The fatty acid 18:4n-3 was a major driver of variability in krill fatty acid composition, with no obvious seasonal driver. This is the first study to report krill fatty acid data during all four seasons over consecutive years. This large-scale study highlights the value of using fisheries samples to examine seasonal and annual fluctuations in krill diet and condition.

Lineage

Maintenance and Update Frequency: notPlanned

Statement: 2.3.1. Sample collection
Krill were caught on board the fishing vessel FV Saga Sea (Aker BioMarine, Oslo, Norway) during their December-September fishing seasons from 2013–2016. The three fishing seasons (December 2013 - September 2014, December 2014 - September 2015, and December 2015 - September 2016) will hereafter be referred to as the three fishing years 2014, 2015, and 2016, respectively. Because FV Saga Sea concluded fishing in mid-September and did not resume until early December every year, spring samples are only from September.
FV Saga Sea fished in FAO statistical subareas 48.1 (West Antarctic Peninsula = WAP), 48.2 (South Orkney Islands = SOI), and 48.3 (South Georgia = SG) within the CCAMLR Convention Area (see Hellessey et al. (2018) for maps of CCAMLR subareas and sampling locations). The vessel predominantly fished at the West Antarctic Peninsula and the South Orkney Islands during summer and autumn, and South Georgia in winter and spring. Fishing locations varied between years as the vessel moved between the CCAMLR areas when the maximum allowable catch for a subarea was reached (48.1), or when sea ice in a subarea made fishing impossible (48.1 and 48.2) or when it attained production goals for a subarea (48.3).
Krill were fished using a continuous catch system whereby a steady (24 h) stream of live krill is pumped from a mid-water trawl net onto the vessel using the vessel’s Eco-Harvesting. (Aker BioMarine) technology. This method of harvesting ensures that krill specimens remain alive and intact.
A fisheries observer took a random sample of twenty krill individuals per day from the catch (there was no selection by size or maturity stage). The sample was divided into two foil or vacuum-sealed packets, lined up in rows of 10 krill per pack. Krill were frozen immediately in a –20°C freezer on board the vessel for 4 h, then transferred to a –80°C freezer for storage. Sample bags were transported on dry ice to Hobart, Tasmania, where they were again stored in a –80°C freezer until needed for analysis.
At least three adult female and three adult male krill 30–60 mm in length were selected from the samples at two-week intervals from December 2013 to September 2016 (391 samples in total). Krill were sexed using a dissecting microscope and weighed (wet mass), and the length of each specimen was measured from the tip of the rostrum to the tip of the uropod using ‘Standard 1’ measurement (Kirkwood 1982). Krill were kept frozen during this process to prevent degradation. A dry mass was obtained later by multiplying the wet mass by 0.2278 to account for the 77.2% water content in krill (Virtue et al. 1993a).

2.3.2 Fatty acid analyses
Whole krill were analysed for fatty acids as the large sample size (N = 391) made it cumbersome to separate out the individual lipid classes (Stübing & Hagen 2003). Specimens were extracted overnight using a modification of the method of Bligh & Dyer (1959), consisting of a methanol:dichloromethane:water (MeOH/CH2Cl2/H2O) solvent mixture (20:10:7 mL). Phase separation was carried out the following day by adding 10 ml CH2Cl2 and 10 ml saline MilliQ H2O, giving a final methanol:dichloromethane:water solvent ratio of 1:1:0.85. The lower layer was drained and the total solvent extract was concentrated using rotary evaporation. The extract was transferred into a pre-weighed 2 ml vial and the solvent was blown down under nitrogen gas to obtain a total lipid extract (TLE) weight. Solvent was added until further procedures were carried out to avoid oxidation.
For fatty acid methyl ester (FAME) preparation, a subsample of the TLE was transferred into a glass test tube fitted with a Teflon-lined screw cap and treated with 3 ml of methylating solution (methanol:dichloromethane: hydrochloric acid, 10:1:1, v:v:v), then heated at 90–100 °C for 1 h 15 min. Samples were cooled and 1 ml of H2O and 1.8 ml of hexane:dichloromethane (4:1, v:v) solution was added to extract the FAME. Samples were then centrifuged for 5 min and the upper layer containing FAME was transferred to a vial, with another 1.8 ml of hexane:dichloromethane then added to the test tube. This process was repeated twice and samples were blown down using nitrogen gas. Samples were made to 1.5 ml with dichloromethane and stored at –20 °C until gas chromatography analysis. Prior to analysis, samples were blown down again using nitrogen gas and 1.5 ml of internal injection standard (23:0 FAME) was added to each vial.
Samples were analysed via gas chromatography using an Agilent Technologies 7890A GC-FID System (Palo Alto, CA, USA) equipped with a non-polar Equity.-1 fused-silica capillary column (15 m length x 0.1 mm internal diameter, 0.1 μm film thickness). Samples (0.2 μl) were injected in splitless mode at an oven temperature of 120 °C with helium as the carrier gas. The oven temperature was raised to 270 °C at a rate of 10 °C per minute, then to 310 °C at 5 °C per minute. Peaks were quantified using Agilent Technologies ChemStation software (Palo Alto, CA, USA) with initial identification based on comparison of retention times with known (Nu Chek Prep mix; http://www.nu-chekprep.com) and fully characterised laboratory (tuna oil) standards. Fatty acid peaks were expressed as a percentage of the total fatty acid area.
Confirmation of component identification was performed by GC-MS of selected samples and was carried out on a Thermo Scientific (Waltham, MA, USA) 1310 GC coupled with a TSQ triple quadruple. Samples were injected using a Tripleplus RSH (Waltham, MA, USA) auto sampler using a non-polar HP-5 Ultra 2 bonded-phase column (50 m length x 0.32 mm internal diameter x 0.17 μm film thickness). The HP-5 column was of similar polarity to the column used for GC analyses. The initial oven temperature of 45 °C was held for 1 min, followed by temperature programming at 30 °C per minute to 140 °C, then at 3 °C per minute to 310 °C, where it was held for 12 min. Helium was used as the carrier gas. Mass-spectrometer operating conditions were as follows: electron impact energy 70 eV; emission current 250 μamp, transfer line 310 °C; source temperature 240 °C; scan rate 0.8 scan/sec and mass range 40–650 Da. Mass spectra were acquired and processed with Thermo Scientific XcaliburTM software (Waltham, MA, USA). Identification and quantification of peaks was conducted using the same standards as GC-FID analysis.

2.3.3. Statistical analyses
Fatty acid quantitative and percentage data for each season were analysed in the RStudio (www.rstudio.com) statistics package (version 0.99.893) using two-way ANOVA, with sex and year as factors. Specific ratios and percentages of fatty acid biomarkers were also analysed using two-way ANOVA with location and year as factors. Two-way ANOVA analyses with season and location as factors were not possible, as data for all seasons were not available for all fishing locations. Type 3 Sums of Squares (SS) were used for statistical analyses when data were unbalanced and Type 1 SS analyses were not appropriate. Data were log- or square-root transformed when they did not meet assumptions of normality. Tukey post-hoc comparisons were used to identify significant differences between factor levels. Data tables are expressed as mean ± standard deviation. For all analyses, α was set at 0.05.
Principal component analyses (PCA) were performed in PRIMER 6 (http://www.primer-e.com) to investigate similarities and differences between groups of fatty acids and identify those that explained most of the variability in the data set. A correlation based PCA (Pearson correlation) was used due to large differences in variances between the fatty acids. Data were transformed using a log (x+1) transformation before analysis, to reduce the influence of fatty acids that had large percentages.