Krill Ocean Acidification Lipid Data

Brief description

Antarctic krill (Euphausia superba) are a keystone species in the Southern Ocean, but little is known about how they will respond to climate change. Ocean acidification, caused by sequestration of carbon dioxide into ocean surface waters (pCO2), is known to alter the lipid biochemistry of some organisms. This can have cascading effects up the food chain. In a year-long laboratory experiment adult krill were exposed to ambient seawater pCO2 levels (400 μatm), elevated pCO2 levels that mimicked near-future ocean acidification (1000, 1500 and 2000 μatm) and an extreme pCO2 level (4000 μatm). The laboratory light regime mimicked the seasonal Southern Ocean photoperiod and krill received a constant food supply. Total lipid mass (mg g -1 DM) of adult krill was unaffected by near-future levels of seawater pCO2. Fatty acid composition (%) and fatty acid ratios associated with immune responses and cell membrane fluidity were also unaffected by near-future pCO2, apart from an increase in 18:3n-3/18:2n-6 ratios in krill in 1500 μatm pCO2 in winter and spring. Extreme pCO2 had no effect on krill lipid biochemistry during summer. During winter and spring, krill in extreme pCO2 had elevated levels of omega-6 fatty acids (up to 1.2% increase in 18:2n-6, up to 0.8% increase in 20:4n-6 and lower 18:3n-3/18:2n-6 and 20:5n-3/20:4n-6 ratios), and showed evidence of increased membrane fluidity (up to three-fold increase in phospholipid/sterol ratios). These results indicate that the lipid biochemistry of adult krill is robust to near-future ocean acidification.

Lineage

Maintenance and Update Frequency: notPlanned

Statement: 5.3.1. Experimental conditions
Experimental conditions are described in detail in Ericson et al. (2018b). Briefly, krill were collected from the Southern Ocean (66-03°S, 59-25°E and 66-33°S, 59-35°E) on the RSV Aurora Australis, using a mid-water trawl net. They were held in shipboard aquaria using standard husbandry methods (see King et al. 2003) and transported to the Australian Antarctic Division Krill Aquarium in Tasmania.
For ocean acidification experiments, five 300L tanks were equilibrated to five pCO2 levels; 400 μatm pCO2 (pH 8.1 control treatment), 1000 μatm pCO2 (pH 7.8), 1500 μatm pCO2 (pH 7.6), 2000 μatm pCO2 (pH 7.4) and 4000 μatm pCO2 (pH 7.1). Seawater temperature of all tanks was held at 0.5ºC (± 0.2). Seawater chemistry for the duration of the experiment is reported in Supplementary Material in Ericson et al. (2018b). Observational units (CO2 treatment tanks) could not be replicated, due to the large tank size required to achieve the best possible animal husbandry for this pelagic species, and the limited space and resources available for these large tanks over such a long-term study. Tanks were inspected daily, and there was no visual evidence to suggest that tank effects were confounding our experimental results.
Two hundred krill were randomly assigned to each tank on the first day of the experiment (25th January 2016), and reared in these pCO2 treatments until the experiment ended on the 12th December 2016. Light was controlled in the laboratory to mimic the seasonal Southern Ocean light regime (66°S, 30m depth) and krill were fed six days per week with a microalgal diet of the Antarctic species Pyramimonas gelidicola (2 x 104 cells mL-1), and Reed Mariculture Inc. (USA) cultures of Thalassiosira weissflogii (8.8 x 103 cells mL-1), Pavlova lutheri (4.5 x 104 cells mL-1) and Isochryisis galbana (5.5 x 10 cells mL-1).

5.3.2. Sample collection and lipid extraction
Krill were sampled from the pCO2 treatment tanks in experimental weeks 1, 2, 4 and 5 (summer), 26 (winter), and 39, 41 and 43 (spring). Five to ten krill were sampled from each tank during each sampling week (only three krill were sampled from the 4000 μatm pCO2 tank due to increased mortality in that tank (see Ericson et al. 2018b) and lower overall numbers of krill). Individual krill were placed in cryo-tubes and frozen immediately at –80°C until needed for lipid analysis.
Krill were weighed (wet mass), and the length of each specimen was measured from the tip of the rostrum to the tip of the uropod using measurement ‘Standard Length 1’ (Kirkwood 1984). To prevent sample degradation, krill were kept frozen during the measuring process. A dry mass (g) for each krill sample was obtained by multiplying the wet mass by 0.2278 to account for the 77.2% water content in krill (Virtue et al. 1993a).
Krill specimens were added to separatory funnels and extracted using a modified Bligh and Dyer (1959) method consisting of a methanol:dichloromethane:water (MeOH:CH2Cl2:H2O) solvent mixture (20:10:7 mL), and overnight extraction. Phase separation was carried out the following day by adding 10 mL CH2Cl2 and 10 mL saline MilliQ H2O to each separatory funnel, giving a final MeOH:CH2Cl2:H2O solvent ratio of 1:1:0.85. The lower layer was drained into a round bottomed flask, and the total solvent extract was concentrated using rotary evaporation. The concentrated extract was transferred into a pre-weighed 2 mL vial and the solvent was blown down under nitrogen (N2) gas to obtain a total lipid extract (TLE) weight. Solvent (CH2Cl2) was added until further procedures were carried out to avoid oxidation.

5.3.3. Lipid class analysis
TLE were used to obtain the lipid class composition of each sample. Aliquots (1μl) of each TLE were spotted on chromarods and developed in a solvent bath of hexane:diethyl-ether:acetic acid (90:10:0.1 mL, v:v:v) for 25 min, before drying in an oven at 50ºC for 10 min. Chromarods were placed in an Iatroscan MK-5 TLC/FID analyser (Iatron Laboratories, Tokyo, Japan) for analysis. A standard solution of known quantities of wax esters (WE), triacylglycerols (TAG), free fatty acids (FFA), sterols (ST), and phospholipids (PL) was used to confirm peak identities and to calibrate the flame ionisation detector. Lipid class peaks were labelled using SIC-480II Iatroscan Integrating Software v.7.0-E, quantified using predetermined linear regressions, and expressed as mg per g of krill dry mass (mg g DM -1). Triacylglycerol data is presented in Ericson et al. (2018b). Only the PL to ST ratio is presented in this manuscript as we were primarily interested in investigating homeoviscous adaptation in krill.

5.3.4. Fatty acid analysis
To prepare fatty acid methyl esters (FAME), a subsample of the TLE was transferred to a glass test tube fitted with a Teflon lined screw cap, and treated with 3 mL methylating solution (MeOH : CH2Cl2 : HCl (hydrochloric acid), 10:1:1, v:v:v). The sample was then heated at 90 – 100°C for 1 hr 15 mins. Samples were cooled and 1 mL of H2O and 1.8 mL of C6H14 (hexane): CH2Cl2 solution was added to extract the FAME. Samples were then centrifuged for five minutes and the upper layer containing FAME was transferred to a vial. An additional 1.8 mL of C6H14:CH2Cl2 was added to the test tube and samples were centrifuged again. This process was repeated three times in total, and samples were blown down using N2 gas in between transfers. FAME samples were made up to 1.5 mL with CH2Cl2 and stored at –20°C until further analysis. Prior to analysis, samples were blown down again using N2 gas and 1.5 mL of internal injection standard (23:0 FAME) was added to each vial.
Samples were analysed via gas chromatography (GC-FID) using an Agilent Technologies 7890A GC System (Palo Alto, California USA) equipped with a non-polar Equity™-1 fused silica capillary column (15 m x 0.1 mm internal diameter and 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. Agilent Technologies ChemStation software was used to quantify fatty acid peaks, with initial identification based on comparison of retention times with known (Nu Chek Prep mix) and laboratory (fully characterised tuna oil) standards. Fatty acid peaks were expressed as a percentage of the total fatty acid area.
Confirmation of component identification was performed by gas chromatography-mass spectrometry (GC-MS) of selected samples and was carried out on a ThermoScientific 1310 GC coupled with a TSQ triple quadruple. Samples were injected using a Tripleplus RSH auto sampler using a non polar HP-5 Ultra 2 bonded-phase column (50 m x 0.32 mm i.d. 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 an increase in temperature of 30°C per minute to 140°C, then at 3°C per minute to 310°C, where it was held for 12 minutes. Helium (He) was used as the carrier gas. The operating conditions of the GC-MS were: 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. Thermo Scientific XcaliburTM software (Waltham, MA, USA) was used to process and acquire mass spectra.
Mean fatty acid chain length (MCL) was calculated using the equation from Bennett et al. (2018):
MCL =  (mg fatty acid g lipid -1 x C) / total mg fatty acid g lipid -1
where C = number of carbon atoms

5.3.5. Statistical analyses
Principal component analyses (PCA) were carried out in PRIMER 6 (http://www.primer-e.com). Pearson correlation was used due to differences in fatty acid variances, and data were transformed (log x+1) before analysis. All other statistical analyses were carried out in RStudio (v 1.1.453; www.rstudio.com). Total lipid, specific fatty acids, lipid class and fatty acid ratios were analysed using Two Way ANOVA with pCO2 and week as main effects, and a pCO2*week interaction term. Tukey comparisons were used to compare levels of pCO2 with one another. On visual assessment of the data, weeks 1 – 5 were analysed as a group, and weeks 26 – 43 were analysed as a separate group, as the groups had heterogeneous variances and represented two distinct data sets. Type 3 Sums of Squares were used as the sampling regime was unbalanced. Log or square root transformations were applied when assumptions of normality and/or homogeneity of variances were not met. For Two Way ANOVA of total lipid data, one outlier was removed from the statistical analysis in order to meet assumptions of homogeneity of variances. Principal component figures were created in PRIMER 6, and all other figures were created using the RStudio packages ggplot2, plyr and dplyr.