Ocean acidification changes the structure of an Antarctic coastal protistan community

Brief description

Experimental Set-up:
An unreplicated, 6-level, dose-response experiment was conducted on a natural microbial community over a range of pCO2 levels (343, 506, 634, 953, 1140 and 1641 micro atm). Seawater was collected on the 19th November 2014 approximately 1 km offshore from Davis Station, Antarctica (68 degrees 35' S, 77 degrees 58' E) from an area of ice-free water amongst broken fast-ice. The seawater was collected using a thoroughly rinsed 720L Bambi bucket slung beneath a helicopter and transferred into a 7000 L polypropalene reservoir tank. Six 650 L polyethene tanks (minicosms), located in a temperature-controlled shipping container, were immediately filled via teflon lined house via gravity with an in-line 200 micron Arkal filter to exclude metazooplankton. The minicosms were simultaneously filled to ensure they contained the same starting community. The ambient water temperature at time of collection was -1.0 degrees C and the minicosms were maintained at a temperature of 0 degrees C plus or minus 0.5 degrees C. At the centre of each minicosm there was an auger shielded for much of its length by a tube of polythene. This auger was rotated at 15 rpm to gently mix the contents of the tanks. Each minicosm tank was covered with an acrylic air-tight lid to prevent pCO2 off-gasing outside of the minicosm headspace.
The minicosm experiment was conducted between the 19th November and the 7th December 2014. Initially, the contents of the tanks were given a day to equibrate to the minicosms. This was followed by a five day acclimation period to increasing pCO2 at low light (0.8 plus or minus 0.2 micro mol m-1 s-1), allowing cell physiology to acclimated to the pCO2 increase (days 1-5). During this period the pCO2 was progressively adjusted over five days to the target level for each tank (343 - 1641 micro atm). Thereafter pCO2 was adjusted daily to maintain the pCO2 level in each treatment (see carbonate chemistry section below). Following acclimation to the various pCO2 treatments light was progressively adjusted to 89 plus or minus 16 micro mol m-2 s-1 at a 19 h light:5 h dark cycle. The community was incubated and allowed to grow for a further 10 days (days 8-18) with target pCO2 adjusted back to target each day (see carbonate chemistry section below).
For a more detailed description of minicosm set-up, lighting and carbonate chemistry see;
Davidson, A. T., McKinlay, J., Westwood, K., Thomson, P. G., van den Enden, R., de Salas, M., Wright, S., Johnson, R., and Berry, K.:Enhanced CO2 concentrations change the structure of Antarctic marine microbial communities, Mar. Ecol. Prog. Ser., 552, 93-113, 2016.
Deppeler, S. L., Petrou, K., Westwood, K., Pearce, I., Pascoe, P., Schulz, K. G., and Davidson, A. T.: Ocean acidification effects on productivity in a coastal Antarctic marine microbial community, Biogeosciences, 2017.

Light microscopy sampling and analysis:
Samples from each minicosm were collected on days 1, 3, 5, 8, 10, 12, 14, 16 and 18 for microscopic analysis to determine protistan identity and abundance. Approximately 960 mL were collected from each tank, on each day. Samples were fixed with 20 40 mL of Lugol's iodine and allowed to sediment out at 4 degrees C for greater than or equal to 4 days. Once cells had settled the supernatant was gently aspirated till approximately 200 mL remained. This was transferred to a 250 mL measuring cylinder, again allowed to settle (as above), and the supernatant gently aspirated. The remaining 20 mL. This final 20 mL was transferred into a 30 mL amber glass bottle.
All samples were stored and transported at 4 degrees C to the Australian Antarctic Division, Hobart, Australia for analysis.
Lugols-fixed and sedimented samples were analysed by light microscopy between July 2015 and February 2017. Between 2 to 10 mL (depending on cell-density) of lugols-concentrated samples was placed into a 10 mL Utermohl cylinder (Hydro-Bios, Keil) and the cells allowed to settle overnight. Due to the large variation in size and taxa, a stratified counting procedure was employed to ensure both accurate identification of small cells and representative counts of larger cells. All cells greater than 20 microns were identified and counted at 20x magnification; those less than 20 microns at 40x magnification. For larger cells (greater than 20 microns), 20 randomly chosen fields of view (FOV) at 3.66 x 106 microns2 counted to gain an average cells per L. For smaller cells (less than 20 microns), 20 randomly chosen FOVs at 2.51 x 105 microns2 were counted. Counts were conducted on an Olympus IX 81 microscope with Nomarski interference optics. Identifications were determined using (Scott and Marchant, 2005) and FESEM images. Autotrophic protists were distinguished from heterotrophs via the presence of chloroplasts and based on their taxonomic identity.

Electron microscopy sampling and analysis:
A further 1 L was taken on days 0, 6, 13 and 18 for analysis by Field Emission Scanning Electron Microscope (FESEM).
25 These samples were concentrated to 5 mL by filtration over a 0.8 micron polycarbonate filter. Cells were resuspended, the concentrate transferred to a glass vial and fixed to a final concentration of 1% EM-grade gluteraldehyde
(ProSciTech Pty Ltd).
All samples were stored and transported at 4 degrees C to the Australian Antarctic Division, Hobart, Australia for analysis.
Gluteraldehyde-fixed samples were prepared for FESEM imaging using a modified polylysine technique (Marchant and Thomas, 30 1983). In brief, a few drops of gluteraldehyde-fixed sample were placed on polylysine coated cover slips and post-fixed with OsO4 (4%) vapour for 30 min, allowing cells to settle onto the coverslips. The coverslips were then rinsed in distilled water and dehydrated through a graded ethanol series ending with emersion in 100% dry acetone before being critically point dried in a Tousimis Autosamdri-815 Critical Point Drier. The coverslips were mounted onto 12.5 mm diameter aluminium stubs and sputter-coated with 7 nm of platinum/palladium in a Cressington 208HRD coater. Imaging of stubs was conducted by JEOL JSM6701F FESEM and protists identified using (Scott and Marchant, 2005).

All units are in cells per L estimates from individual field of view counts (FOV)

Protistan taxa and functional group descriptions and abbreviations:
Autotrophic Dinoflagellate (AD) - including Gymnodinium sp., Heterocapsa and other unidentified autotrophic dinoflagellates
Bicosta antennigera (Ba)
Chaetoceros (Cha) - mainly Chaetoceros castracanei and Chaetoceros tortissimus but also other Chaetoceros present including C. aequatorialis var antarcticus, C. cf. criophilus, C. curvisetus, C. dichaeta, C. flexuosus, C. neogracilis, C. simplex
Choanoflagellates (except Bicosta) (Cho) - mainly Diaphanoeca multiannulata but also Parvicorbicula circularis and Parvicorbicula socialis present in low numbers
Ciliates (Cil) - mostly cf. Strombidium but other ciliates also present
Discoid Centric Diatoms greater than 40 microns (DC.l) - unidentified centrics of the genera Thalassiosira, Landeria, Stellarima or similar
Discoid Centric Diatoms 20 to 40 microns (DC.m) - unidentified centrics of the genera Thalassiosira, Landeria, Stellarima or similar
Discoid Centric Diatoms less than 20 microns (DC.s) - unidentified centrics of the genera Thalassiosira
Euglenoid (Eu) - unidentified
Fragilariopsis greater than 20 microns (F.l) - mainly Fragilariopsis cylindrus, some Fragilariopsis kerguelensis and potentially some Fragilariopsis curta present in very low numbers
Fragilariopsis less than 20 microns (F.s) - mainly Fragilariopsis cylindrus, and potentially some Fragilariopsis curta present in very low numbers
Heterotrophic Dinoflagellates (HD) - including Gyrodinium glaciale, Gyrodinium lachryma, other Gyrodinium sp., Protoperidinium cf. antarcticum and other unidentified heterotrophic dinoflagellates
Landeria annulata (La)
Other Centric Diatoms (OC) - Corethronb pennatum, Dactyliosolen tenuijuntus, Eucampia antarctica var recta, Rhizosolenia imbricata and other Rhizosolenia sp.
Odontella (Od) - Odontella weissflogii and Odontella litigiosa
Other Flagellates (OF) - Dictyocha speculum, Chrysochromulina sp., unknown haptophyte, Phaeocystis antarctica (flagellate and gamete forms), Mantoniella sp., Pryaminmonas gelidicola, Triparma columaceae, Triparma laevis subsp ramispina, Geminigera sp., Bodo sp., Leuocryptos sp., Polytoma sp., cf. Protaspis, Telonema antarctica, Thaumatomastix sp. and other unidentified nano- and picoplankton
Other Pennate Diatoms (OP) - Entomonei kjellmanii var kjellmanii, Navicula gelida var parvula, Nitzschia longissima, other Nitzschia sp., Plagiotropus gaussi, Pseudonitzschia prolongatoides, Synedropsis sp.
Phaeocystis antarctica (Pa) - colonial form only
Proboscia truncata (Pro)
Pseudonitzschia subcurvata (Ps)
Pseudonitzschia turgiduloies (Pt)
Stellarima microtrias (Sm)
Thalassiosira antarctica (Ta)
Thalassiosira ritscheri (Tr)
*.se = standard error for mean cell per L estimate ie. Tr.se = standard error for the mean cells per L for Thalassiosira ritscheri based on individual FOV estimates as described in methods above.

Davis Station Antarctica
Experiment conducted between 19th November and 7th December 2014.

Lineage

Progress Code: completed

Statement: This is based on unreplicated data, standard errors are calculated based on microscope field of view pseudoreplicates.
The following functional groups were present in very low numbers and therefore there was high variance between counts resulting in high standard error of mean cells per litre estimates;
Autotrophic and heterotrophic dinoflagellates, ciliates, other flagellates, discoid centric diatoms (greater than 40, 20 to 40 and less than 20 microns), other centric and pennate diatoms.
All other taxa and functional groups have low variance on counts and are statistically viable.

Notes

Purpose
The Southern Ocean is particularly vulnerable to ocean acidification due to its cold temperatures, extensive upwelling and naturally large seasonal fluctuations in pH. Microbes are an important component of these waters through the roles they play in driving productivity, elemental cycles and ocean biogeochemistry, meaning their response to environmental stressors is a key determinant of Southern Ocean feedbacks to global climate change (Arrigo and Thomas, 2004; Arrigo et al., 2008; Kirchman, 2008). Despite their importance, relatively little is known about the sensitivity of Antarctica marine microbes to ocean acidification and this limits our ability to predict the how the Southern Ocean will be impacted in the future, and the feedback this may have on global climate change. This study will assessed the following questions on a natural microbial community from nearshore East Antarctic waters. 1. Is there a change in protistan community composition and abundance with increased concentration of pCO2? And what pCO2 concentration elicits this change? 2. Does an acclimation period allow the protistan community the cope to increased concentrations of pCO2? 3. Is the effect of increased pCO2 concentration species-specific? And if it is, what are the potential driving mechanisms behind these responses? 4. Does the results of this minicosm experiment agree with those of previously conducted experiments at this site by Davidson et al. (2016) and Thomson et al. (2016) in the austral summer of 2008-09? Therefore is the response of the protistan community consistent both across a season and between a season at Prydz Bay, East Antarctica?