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Experimental Design A six-level, dose-response ocean acidification experiment was run on a natural microbial community from nearshore Antarctica, between 19th November and 7th December 2014. Seawater was collected from approximately 1 km offshore of Davis Station, Antarctica (68◦ 35’ S, 77◦ 58’ E), pre-filtered (200 μm), and transferred into six 650 L tanks (minicosms) located in a temperature-controlled shipping container. Six CO2 levels were achieved by altering the fugacity of carbon dioxide (ƒCO2) within each minicosms. The ƒCO2 was adjusted stepwise to the target concentrations for each minicosm (343, 506, 634, 953, 1140, 1641 μatm) over a five-day period using 0.2 μm filtered seawater enriched with CO2. This acclimation to CO2 was conducted at low light (0.9 ± 0.2 μmol m−2 s−1) so there was low growth of the phytoplankton. Light levels were then increased over a further two days to 90.52 ± 21.45 μmol m−2 on a 19:5 light/dark non-limiting light cycle. After this acclimation period, the microbial community was allowed to grow for 10 days (days 8-18), during which the ƒCO2 levels within each minicosm was adjusted daily to maintain the target ƒCO2 level for each minicosm, and light levels were kept constant. No nutrients were added during the experiment. 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, 15(1), 2018. Sample Collection Samples of 40-400 L were collected and sequentially size-fractionated filtered onto 293 mm biomass filters with 3.0 and 0.1 μm pore-sized polyethersulfone membrane filters (Pall XE20206 Disc 3.0 μm Versapor 293 mm and 656552 Disc 0.1 μm Supor 293 mm) using the design of the Global Ocean Sampling expedition (Rusch et al., 2007). Samples were collected on days 0 (immediately after seawater collection), 12 (mid-exponential growth) and 18 (end of experiment). On day 0, 400 L of seawater was collected from the reservoir tank (pre-filtered 200 μm), from which all the minicosms were filled, to allow characterisation of the initial community. This sample was collected from the reservoir, and not the minicosms, due to the large volume needed to collect sufficient microbial biomass on the filters. On day 12 and 18, 40 L was collected from each minicosm for filtration. The later samples were of a smaller volume due to the increase in biomass in the minicosms during the experiment, meaning less volume of water was required to gain sufficient material on the filters to perform molecular analysis. The filter membranes containing the concentrated microbial biomass were stored in 15 mL of storage buffer, flash frozen in liquid nitrogen and stored at - 80◦C. The storage buffer was freshly prepared on each sampling day with a mixture of 2.5 mM EGTA, 2.5 mM EDTA, 0.1 mM Tris-EDTA, RNA Later (0.5x house prepared), 1 mM PMSF and Protease Inhibitor Cocktail VI (Ng et al., 2010). Between samples the filtration apparatus was sequentially washed with 2 x 25 L 0.1 M NaOH, 2 x 25 L 0.07% Ca(OCl)2 and 2 x 25 L fresh water. All samples were stored and transported at -80◦C to the Australian Antarctic Division, Hobart, Australia for DNA extraction. DNA Extraction and Sequencing The DNA was extracted from half of each filter (3.0 and 0.1 μatm per sample) via the method described in Rusch et al. (2007). In short, the filters were cut into small pieces and agitated in a lysozyme and sucrose buffer for 60 minutes and underwent three freeze/thaw cycles in a Proteinase K solution. This was followed by a gentler agitation at 55◦C for 2 hrs to remove all contents from the filter membranes. DNA was then separated using buffer saturated phenol, pelleted and washed in alcohol. The final DNA pellet was dissolved and stored in a 3 M sodium acetate (pH 8.0) and 100% ethanol solution and stored at - 80◦C. The DNA was transported and stored at 4◦C to the University of Queensland, St Lucia, Australia for sequencing within two months of extraction. Eukaryotic 18S rRNA genes (V8-V9 regions) were amplified using polymerase chain reaction (PCR) with the primers V8f (5’ - AT AAC AGG TCT GTG ATG CCC T - ’3) and 1510r (5’ - CCT TCY GCA GGT TCA CCT AC - ’3) (Bradley, 2016). The 16S rRNA genes V8 region were amplified using PCR and primers 926F (5’-AAA CTY AAA KGA ATT GAC GG-3’) and 1392wR (5’-ACG GGC GGT GTG RC-3’) (Engelbrektson et al., 2010). PCR was performed using 1 or 1.5 μL of sample DNA, 2.5 μL 1x PCR buffer minus Mg+2 (Invitrogen), 0.75 μL MgCl2, 0.5 μL deoxynucleoside triphosphate (dNTPs, Invitrogen), 0.125 μL U Taq DNA Polymerase (Invitrogen), 0.625 μL of forward/reverse primer and made up to the final volume of 25 μL using molecular biology grade water. Forward and reverse primers were modified at the 5’-end to contain an Illumina overhang adaptor with P5 and i7 Nextera XT indices, respectively. The PCR thermocycling conditions were as follows: 94◦C for 3 min, 35 cycles of 94◦C for 45 sec, 55◦C for 30 sec, 7◦C for 10 min and a final extension of 72◦C for 10 min. Amplifications were performed using a Vertiti®96-well Thermocycler (Applied Biosystems) and success, amplicon size and quality was determined by gel electrophoresis. The resultant amplicons were purified using Agencourt AMPure magnetic beads (Axygen Biosciences), dual indexed using Nextera XT Index Kit (Illumina). The indexed amplicons were purified using Agencourt AMPure XP beads and quantified using PicoGreen dsDNA Quantification Kit (Invitrogen). Equal concentrations of each sample were pooled and sequenced on an Illumina MiSeq at the University of Queensland’s School for Earth and Environmental Science using 30% PhiX Control v3 (Illumina) and a MiSeq Reagent Kit v3 (600 cycle; Illumina). Bioinformatics Sequencing data and runs were merged to produced single FASTQ file for 16S and 18S rDNA per sample and imported in QIIME2 (v2019.9) (Caporaso et al., 2010). A modified version of the UPARSE analysis pipeline was used to analyse the data. Specifically, the primer sequences were removed from forward reads of the 16S rDNA and reverse complement of the 18S rDNA Illumina read pairs, and chimeras removed using UCHIME2 (Edgar, 2016). These were then trimmed to a length of 200 bp and high-quality sequences identified using USEARCH (v10.0.240) (Edgar, 2010). Duplicate sequences were removed and a set of unique operational taxonomic units (OTUs) were generated using USEARCH employing a 97% OTU similarity radius. Mitochondrial and chloroplast OTUs were classified and removed from the 16S rDNA sequence data using the BIOM tool suite (McDonald et al., 2012). Representative OTU sequences were assigned taxonomy using SILVA132 (Quast et al., 2012) and PR2 (Guillou et al., 2012) for the eukaryotic group Bacillariophyceae (diatoms). Taxonomic assignments were validated against microscopy identifications conducted on the same samples (Chapter 3, Hancock et al. 2018) as well as phylogenetic trees built in iTOL (Letunic and Bork, 2006). Residual eukaryotic chloroplast and mitochondrial sequences were removed from the 16S rDNA data. Other obvious contaminants were removed manually including: Escherichia-Shigella (16S rDNA OTU75) and Saccharomycetales (18S rDNA OTU7, 146 and 160). Escherichia-shigella was removed as this group likely represents external contamination, similarly Saccharomycetales are yeast and are obvious skin-driven contaminants. A total of 9448 OTUs were identified from the 16S rDNA reads and 232 OTUs from the 18S rDNA read data. The number of reads were rarefied to 1300 and 1200 reads per sample for the 18S and 16S rDNA datasets respectively. The following samples were removed due to lack of extracted, amplified and/or sequenced DNA, or due to low quality reads and/or low read numbers: 18S, 3.0 μm, day 18, 634 μatm ƒCO2 treatment 18S, 0.1 μm, day 12, 343 μatm or control ƒCO2 treatment 18S, 0.1 μm, day 18, 343 μatm or control ƒCO2 treatment 16S, 0.1 μm, day 18, 506 μatm ƒCO2 treatment Statistical Analysis The minicosm experiment was based on a repeated measure design, therefore due to being a dose-response experiment with no replication, no formal statistics could be undertaken on the interactions between time and ƒCO2. The richness (number of taxa) and evenness (equivalent to abundances within a sample) of the eukaryotic and prokaryotic microbial communities within each minicosm over time was estimated using three different alpha diversity indexes: observed number of OTUs (Sobs) (DeSantis et al., 2006), the Chao1 estimator of richness (Colwell et al., 2004), and Simpson’s diversity index and Berger-Parker index which account for both richness and evenness (Simpson, 1949; Berger and Parker, 1970) using QIIME2. Clustering and ordinations were performed on Bray-Curtis resemblance matrices of the rarefied, square-root transformed OTU data as per Chapter 3 (Hancock et al., 2018). In brief, hierarchical agglomerative cluster analyses were performed using group-average linkage, and significantly different clusters were determined using similarity profile permutations method (SIMPROF) (Clarke et al., 2008). Both unconstrained (non-metric multidimensional scaling, nMDS) and constrained (canonical analysis of principal coordinates, CAP) ordinations were performed using the Bray-Curtis resemblance matrixes (Kruskal, 1964a,b; Oksanen et al., 2017). The constraining variables in the CAP analysis were ƒCO2, Si, P and NOx. All cluster and ordination analyses were performed using R v.1.1.453 (R Core Team, 2016) and the add-on package Vegan v.2.5-3 (Oksanen et al., 2017). A full description of the statistical methods used for this paper is described in; Hancock, A. M., Davidson, A. T., McKinlay, J., McMinn, A., Schulz, K. G., and van den Enden, R. L. Ocean acidification changes the structure of an Antarctic coastal protistan community, Biogeosciences, 15(1), 2018.
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.
An unreplicated, six-level dose-response experiment was conducted using 650 L incubation tanks (minicosms) adjusted to fugacity of carbon dioxide (fCO2) from 343 to 11641 uatm. The minicosms were filled with near-shore water from Prydz Bay, East Antarctica and the protistan composition and abundance was determined by microscopy analysis of samples collected during the 18 day incubation. Abundant taxa with low variance were examined separately, but rare taxa with high variance were combined into functional groups (descriptions below). Cluster analyses and ordinations were performed on Bray-Curtis resemblance matrixes formed from square-root transformated abundance data. This transformation was assessed as appropriate for reducing the influence of abundance species, as judged from a one-to-one relationship between observed dissimilarities and ordination distances (ie. Shepard diagram, not shown). The Bray-Curtis metric was used as it is recommended for ecological data due to its treatment of joint absences (ie. these do not contribute towards similarity), and giving more weight to abundant taxa rather than rare taxa. The data days 1 to 8 and then days 8 to 18 were analysed separately to distinguish community structure in the acclimation period and in the exponential growth phase during the incubation period of the experiment. Hierarchical agglomerative cluster analyses, based on the Bray-Curtis resemblance matrix, was performed using group-average linkage. Significantly different clusters of samples were determined using SIMPROF (similarity profile permutations method) with an alpha value of 0.05 and based on 1000 permutations. An unconstrained ordination by non-metric multidimensional scaling (nMDS) was performed on the resemblance matrix with a primary (`weak') treatment of ties. This was repeated over 50 random starts to ensure a globally optimal solution according to . Clusters are displayed in the nMDS using colour. Weighted average of sample scores are shown in the nMDS to show the approximate contribution of each species to each sample. The assumption of a linear trend for predictors within the ordination was checked for each covariate, and in all instances was found to be justified. A constrained canonical analysis of principal coordinates (CAP) was conducted according to the Vegan protocol using the Bray-Curtis resemblance matrix. This analysis was used to assess the significance of the environmental covariates, or constraints, in determining the microbial community structure. Unlike the nMDS ordination, the CAP analysis uses the resemblance matrix to partition the total variance in the community composition into unconstrained and constrained components, with the latter comprising only the variation that can be attributed to the constraining variables, fCO2, Si, P and NOx. Random reassignment of sample resemblance was performed over 199 permutations to compute the pseudo-F statistic as a measure of significance of each environmental constraint in the structural change of the microbial community. A forward selection strategy was used to choose a minimum subset of significant constraints that still account for the majority of the variation within the microbial community. All analysis were performed using R v1.0.136 and the add-on package vegan v2.4-2. 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.
This metadata record contains the results from 11 bioassays conducted with 2 species of Antarctic marine microalgae. Seven tests were conducted with Phaeocystis antarctica (Prymnesiophyceae), assessing the toxicity of copper, cadmium, lead, zinc and nickel. Four tests were conducted with Cryothecomonas armigera (Incertae sedis), assessing the toxicity of copper only. Test conditions for both algae are described in the excel spreadsheets. In summary, tests for P. antarctica and C.armigera, were carried out at 0 plus or minus 2 degrees C, 20:4 h light:dark (150-200 micro mol/m2/s, cool white 36W/840 globes), in natural filtered (0.45 microns for P.antarctica and 0.22 microns filtered for C. armigera) seawater (salinity - 35 ppt, pH - 8.1 plus or minus 0.2). For both species, filtered seawater was supplemented with 1.5 mg/L NO3- and 0.15 mg/L of PO43-. All tests were carried out in silanised 250-mL glass flasks, with glass lids. Test volumes for P.antartica and C.armigera were 50 mL and 80 mL, respectively. All tests consisted of 3-5 metal treatments, with 3 replicates per treatment, alongside 3 replicate controls (natural filtered seawater). Seawater was spiked with metal solutions to achieve required concentration. Concentrations tested are recorded in excel datasheets. The following replicate toxicity tests were completed for P. antarctica: - 5 tests with copper (1-20 micro g/L) - 4 tests with lead (10-500 micro g/L) - 3 tests with cadmium (100-2000 micro g/L) - 3 tests with zinc (100-2000 micro g/L) - 3 tests with nickel (200-1000 micro g/L) For C. armigera, 1 rangefinder test was carried out testing 6 concentrations (1-100 micro g/L), and 3 definitive test, with 5 concentrations (15-100 micro g/L). The age of P. antarctica and C.armigera at test commencement was 8-12 days, and 25-30 days, respectively. Algal cells were centrifuged and washed to remove nutrient rich media, and test flasks were inoculated with between 1-3 x103 cells/mL. Cell densities in all toxicity tests were determined by flow cytometry. The flow cytometer was also used to simultaneously measure change sin chlorophyll a fluorescence intensity, cell size and internal cell granularity. Toxicity tests were continued until cell densities in the control treatments had increased 16-fold. Toxicity tests with P. antarctica were carried out over 10 days, with cell densities in each replicate flask measured every 2 days. Toxicity tests with C. armigera were carried out over 23-24 days, with cell densities determined twice a week. The growth rate (cell division; u) was calculated as the slope of the regression line from a plot of log10 (cell density) versus time (h). Growth rates for all treatments were expressed as a percentage of the control growth rates. The pH in all treatments was measured on the first and last day of the test, as well as on day 6 for P. antarctica tests and an additional two times per week for C. armigera tests. Sub-samples (5 mL) for analysis of dissolved metal concentrations were taken from each treatment on days 0, 6 and 10 for P. antarctica tests, and on days 0, 7, 14, 21, and 24 for C. armigera tests. Sub-samples were filtered through an acid washed (10% HNO3, Merck) 0.45-micron membrane filter and syringe, and acidified to 0.2% with Tracepur nitric acid (Merck). All toxicity test results were calculated using measured dissolved metal concentrations, which were determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES) for Cu, Cd, Pb, Ni and Zn and using inductively coupled plasma-mass spectrometry (ICP-MS; Agilent 7500CE) for lowest concentration Cu samples (nominal concentration 1 micro g/L). Detection limits for Cu, Cd, Pb, Ni and Zn were 1, 0.12, 1.7, 1.2 and 0.1 micro g/L, respectively (ICP-AES) and 0.05 micro g/L (ICP-MS) for low concentration Cu samples. The specific growth rates (u) and corresponding measured metal concentrations were used to calculate toxicity test values using Toxcalc (Version 5.0.23, TidePool Scientific Software, San Francisco, CA, USA). Data were tested for normal distribution using Shapiro-Wilk's test (p greater than 0.01); and equal variances using Bartlett's test (p = 0.09). The inhibitory concentration which reduced population growth rate by x% (ICx) compared to controls was calculated using linear interpolation. The Dunnett's multiple comparison test was used to determine which treatments were significantly different to the control (2 tailed, p less than or equal to 0.05), and to calculate the no observable effect concentration (NOEC) and the lowest observable effect concentration (LOEC). Data for each toxicity test are provided in individual excel spreadsheets, identified by the species tested, the test number for that species and the date the test started. A summary table of details for the 11 tests is provided in the file: Summary table.xlsx. The first worksheet for each test file is titled "Test Conditions". This sheet provides information on the toxicity test e.g. species and metals tested, dates, test conditions, as well as explanation of abbreviations, definitions of toxicity values etc. The second worksheet includes the raw cell densities determined in each flask, the calculated growth rates, and the measured pH and metal concentrations. For C. armigera data sheets, there is an additional worksheet, "Measured Cu and pH" which includes all measured pH values and metal concentrations across the 24-day period. Following the growth rate sheets are the statistical outputs for each metal, which were all generated using Toxcalc. Finally, if additional cellular parameters were measured (Chlorophyll a fluorescence, cell size and internal cell granularity), the raw data for each parameter is include in a worksheet, "Metal cellular parameters". Data were collected in an Australian laboratory (CSIRO Land and Water, Centre for Environmental Contaminants Research, Lucas Heights, 2234, NSW) during May 2013 - April 2014. The tests used microalgal strains that had been previously collected from the Southern Ocean and are cultured within the microalgal collection at the Australian Antarctic Division (AAD). Daughter daughter cultures were transferred to CSIRO, where they were cultured for this work.
This data describes the cellular metal concentrations of Phaeocystis antarctica and Cryothecomonas armigera following exposure to metals singly and in mixtures in laboratory studies. Microalgae were cultured in 80 mL of filtered (less than 0.45 um) seawater and low concentrations of nutrients supplemented with metal stocks to give a range of single and mixture exposures to the metals cadmium, copper, nickel, lead, and zinc. The cellular accumulation and partitioning are used to explain the metal's toxicity (cellular metal fractions are compared to the toxicity data provided in 10.4225/15/5ae93ff723ff8) and assess the risk bioaccumulation of metals to Antarctic marine microalgae may pose in the Southern Ocean food web.
This study assessed the performance of diffusive gradients in thin-films (DGT) with a binding resin that used Chelex-100 (iminodiacetic acid functional groups) to measure cadmium, copper, nickel, lead, and zinc contaminants in Antarctic marine conditions. To do this, three sets of experiments were done: (I) the uptake of metals to DGT samplers was assessed over time when deployed to three metal mixtures of known concentrations (DGT performance page). This allowed for the determination of metal diffusion coefficients in Antarctic marine conditions and demonstrated when metal competition for binding sites were likely to occur. (II) the DGT were deployed in the presence of the microalga Phaeocystis antarctica at a concentration of 1000-3000 cells/mL to investigate how environmentally realistic concentrations of an Antarctic marine microalgae affect the uptake of metals (DGT uptake with algae page). Finally, the DGT-labile concentrations from part (II) were used in reference toxicity mixture models to predict toxicity to the microalgae so they could be compared to a previous study that investigated the toxicity of metal mixtures to Phaeocystis antarctica and Cryothecomonas armigera (DGT toxicity modelling page).
This metadata record contains observed and predicted toxicity data from bioassays with two species of Antarctic marine microalgae: Phaeocystis antarctica (Prymnesiophyceae) and Cryothecomonas armigera (Cercoza). Bioassay exposures were of mixtures of 5 metals at two ratios, an Environmental (ENV) and Equitoxic (EC) mixture. The measured dissolved metal concentrations were used in two mixture reference models, Independent Action (IA) and Concentration Addition (CA), to predict toxicity as population growth rate inhibition. A Flow Cytometer (BD-FACSVerse) was used to measure the density of microalgae over time, which was then converted to a growth rate. An inductively coupled plasma-atomic emission spectrometry (ICP-AES; Varian 730-ES), was used to measure metal concentrations. Data for each microalga is provided in individual excel spreadsheets, identified by the species tested. A word document is provided that contains the R code used to predict toxicity to the two microalgae by the reference models Independent Action and Concentration Addition. The R code also includes the steps required to extend the models to include a deviation parameter “a” that allows for departure from model additivity. A nested F-test then tests for significance between the fit of each test to observed toxicities. This R code has been adapted to use EC10 as parameter estimates, rather than EC50s. The code was adapted from the approach outlined in Hochmuth, J. D.; Asselman, J.; De, S. Are Interactive Effects of Harmful Algal Blooms and Copper Pollution a Concern for Water Quality Management? Water Res. 2014, 60, 41–53. DOI: 10.1016/j.watres.2014.03.041. Single-metal toxicity data and experimental protocols for P. antarctica from the following paper: and C. armigera used in this study can be found in the following papers: A robust bioassay to assess the toxicity of metals to the Antarctic marine microalga Phaeocyctis antarctica. Francesca Gissi, Merrin S. Adams, Catherine K. King, Dianne F. Jolley (2015). Environmental Toxicology and Chemistry. 2015 Feb 20. doi: 10.1002/etc.2949. Chronic toxicity of five metals to the polar marine microalga Cryothecomonas armigera – Application of a new bioassay. Darren J. Koppel, Francesca Gissi, Merrin S. Adams, Catherine K. King, and Dianne F. Jolley, (2017). Environmental Pollution, Volume 228, 2017, Pages 211-221, doi.org/10.1016/j.envpol.2017.05.034.
This metadata record covers ASAC projects 113, 191 and 625. (ASAC_113, ASAC_191, ASAC_625). The total lipid, fatty acid, sterol and pigment composition of water column particulates collected near the Australian Antarctic Base, Davis Station, were analysed over five summer seasons (1988-93) using capillary GC, GC-MS, TLC-FID and HPLC. Polar lipids were the dominant lipid class. Maximum lipid concentrations usually occurred in samples collected in December and January and corresponded with increased algal biomass. Both lipid profiles and microscopic observations showed significant variation in algal biomass and community structure in the water column during each season and on an interannual basis. During the period of diatom blooms (predominantly Nitzschia species) the dominant sterol and fatty acid were trans-22-dehydrocholesterol and 20:5w3, accompanied by a high 16:1w7 to 16:0 ratio. Very high polyunsaturated fatty acid and total lipid concentrations were associated with diatom blooms in the area. Bacterial markers increased late in all seasons after the summer algal blooms. Long chain C30 sterols also increased during the latter half of all seasons. Fjord samples collected in the area reflected greater biomass and diversity in algal and bacterial makers than coastal sites. Signature lipids for the alga Phaeocystis pouchetii, thought to be a major alga in Antarctic waters, were identified in field samples over the five summer seasons studied. Methods Study site Davis Base is situated on the Vestfold Hills, Antarctica and incorporates numerous lakes and fjords (Fig. 1). Samples of water column particulate matter were collected during five summer seasons (1988-93), 500 meters off-shore from Magnetic Island, situated 5 km NW of Davis. Three other sampling areas were situated in the fjords of the Vestfold hills and include two sites in Ellis Fjord, one midway along Ellis Fjord and one near Ellis Fjord mouth and one sample midway along Long Fjord (Fig. 1). These fjords are protected from the marine environment, but are both marine fjords. Davis Station and Magnetic Island were used for the weekly sample sites. The mouth of Long Fjord, the mouth of Ellis Fjord, midway down Long Fjord, the deep basin in Ellis Fjord, O'Gorman Rocks and Hawker island (ocean side) were used for monthly samples. Field collection There was an initial pilot season in 1988-89, which was followed by two more detailed studies in the summers of 1989-90 and 1990-91. Four samples was also analysed from the 1991-92 and five from the 1992-93 summer seasons. During the initial pilot study at Magnetic Island in the 1988-89 summer, three water column particle samples were taken for lipid analyses. The 1989-90 and 1990-91 summer field seasons incorporated weekly sampling of the water column particulates at Magnetic Island. The phytoplankton in the fjords were studied during the summers of 1989-90 and 1990-91. The three sites that were chosen were all sampled three times in each season. Samples were also collected during the 1989-90 and 1990-91 seasons from the Magnetic Island and Fjord site s for pigment analyses. Three and five samples were collected respectively in the 1991-92 and 1992-93 seasons. Samples were also taken for microscopic analyses. For lipid analyses 30-40 liter water column particulate samples were collected at a depth of 10 m. A Seastar or INFILTREX water sampler was used in situ to filter the water through a 14.2 cm Schleicher and Schuell glass fibre filter over a three to four hour period. All filters used during sampling were preheated in a muffle furnace at 500 degrees C overnight to minimise contamination. For pigment analyses 2 to 4 litres were filtered through glass fibre filters (4.7 cm GF/F, nominal pore size 0.7 micro meters). The samples were frozen at -20 degrees C until extraction.
From the abstract of one of the papers: Phytoplankton biomass and speciation were monitored at an inshore site near Davis Station, East Antarctica during three consecutive summer seasons (December-February, 1992-5). Four distinct phytoplankton assemblages were identified in which the dominant species were: Phaeocystis sp., an undescribed Cryptomonas species, Thalassiosira dichotomica, and a mixed assemblage containing Fragilariopsis spp. and Nitzschia spp. Little interannual consistency was found in either the timing of the appearance or disappearance of the various assemblages. Similarly, the seasonal trends in biomass varied dramatically from year to year. Variations in the phytoplankton community can be ascribed, to some extent, to the random variation in a number of factors, including the date of fast ice break out, water column stratification, temperature and salinity, zooplankton grazing and strong winds. Periods of strong wind result in the introduction of offshore or deeper water masses into the shallow inshore environment, where the physical and chemical conditions allow blooms to develop. A number of the papers listed in the reference section are available as pdf's in the download section.
From the abstract of some of the papers: It has been suggested that increased springtime UVB radiation caused by stratospheric ozone depletion is likely to reduce primary production and induce changes in the species composition of Antarctic marine phytoplankton. Experiments conducted at Arthur Harbour in the Antarctic Peninsula revealed a reduction in primary productivity at both ambient and increased levels of UVB. Laboratory studies have shown that most species in culture are sensitive to high UVB levels, although the level at which either growth or photosynthesis is inhibited is variable. Stratospheric ozone depletion, with resultant increased springtime UVB irradiance, has been occurring with increasing severity since the late 1970's. Thus the phytoplankton community has already experienced about 20 years' exposure to increasing levels of UVB radiation. Here we present analyses of diatom assemblages from high-resolution stratigraphic sequences from anoxic basins in fjords of the Vestfold HIlls, Antarctica. We find that compositional changes in the diatom component of the phytoplankton community over the past 20 years cannot be distinguished from long-term natural variability, although there is some indication of a decline in the production of some sea-ice diatoms. We anticipate that our results are applicable to other Antarctic coastal regions, where thick ice cover and the timing of the phytoplankton bloom protect the phytoplankton from the effects of increased UVB radiation. Growth rate, survival, and stimulation of the production of UV-B (280 to 320 nm) absorbing compounds were investigated in cultures of five commonly occurring Antarctic marine diatoms exposed to a range of UV-B irradiances. Experimental UV-B exposures ranged from 20 to 650% of the measured peak surface irradiance at an Antarctic coastal site (0.533 J per square metre per second). The five diatom species (Nitzschia lecointei, Proboscia alata, P. inermis, Thalassiosira tumida and Stellarima microtrias) appear capable of surviving two to four times this irradiance. In contrast to Phaeocystis cf. pouchetti, another major component of the Antarctic phytoplankton, the concentrations of pigments with discrete UV absorption peaks in diatoms were low and did not change significantly under increasing UV-B irradiance. Absorbance of UV-B by cells from which pigments had been extracted commonly exceeded that of the pigments themselves. Most of this absorbance was due to oxidisable cell contents, with the frustule providing the remainder. Survival of diatoms did not correlate with absorption by either pigments, frustules or oxidisable cell contents, indicating that their survival under elevated UV-B irradiances results from processes other than screening mechanisms. Springtime UV-B levels have been increasing in Antarctic marine ecosystems since the 1970's. Effects on natural phytoplankton and sea-ice algal communities, however, remain unresolved. At the Marginal Ice Edge Zone, enhanced springtime UV-B levels coincide with a shallow, stratified water column and a major phytoplankton bloom. In these areas it is possible that phytoplankton growth and survival is adversely impacted by enhanced UV-B. In coastal areas, however, the sea ice, which attenuates most of the UV-B before it reaches the water column, remains until December/January, by which time UV-B levels have returned to long-term seasonal averages. Phytoplankton from these areas are unlikely to show long-term changes resulting from the hole in the ozone layer. Fjords of the Vestfold Hills, eastern Antarctica, have anoxic basins which contain high-resolution, unbioturbated sedimentary sequences. Diatom assemblages from these sequences reflect the diatom component of the phytoplankton and sea-ice algal assemblages at the time of deposition. Twenty-year records from these sequences show no consistent record of change in species composition, diversity or species richness. Six-hundred-year records from the same area also show changes in species abundance greater than those seen in the last 20 years. From these records it can be seen that recent changes in diatom abundances generally fall within the limits of natural variability and there is little evidence of recent changes that might be associated with UV-B-induced change.