This metadata record contains an Excel spreadsheet with Operational Taxonomic Units (OTUs) gained from 16S rRNA gene sequencing of prokaryotes sampled from Biofilm slides deployed as part of the antFOCE experiment in the austral summer of 2014/15 at Casey station, East Antarctica. Refer to antFOCE report section 4.5.3 for deployment, sampling and analysis details. https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 Sampling design 2 trays of 8 horizontal standard glass microscope slides (72 x 25 mm) per chamber. Four of the glass slides were scored with a diamond pencil approximately 18 mm from the right hand end of the slide and deployed scored side up. The remaining four slides were unmodified. Slides were sampled at: * Tmid - one tray per chamber / open plot. The sampled try was repopulated with fresh slides and redeployed * Tend – 2 slides trays per chamber / open plot. Sampling procedure After 31 days deployment, 1 slide tray per chamber / open plot was sampled. At Tend both trays in each chamber / open plot were sampled. To minimize disturbance while being raised to the surface, each tray was removed from the tray holder by divers and placed in a seawater filled container with a lid. On the surface, slides were removed from the tray using ethanol sterilized forceps. The four unscoured slides per chamber / open plot were placed in a plastic microscope slide holder with a sealable lid. The scoured slides were placed individually in 70 ml plastic sample jars. Lab procedure - Casey The slide holder (4 unscoured slides) from each chamber / open plot was frozen at -20C immediately upon return to the lab. The scoured slides were preserved in sea water containing 1% final concentration glutaraldehyde in separate jars. Preservation Issue: Scoured slides were not refrigerated, either at Casey, during RTA or in Kingston before the 26th Nov 2015, when they were transferred to the 4C Cold Store. antFOCE Background The antFOCE experimental system was deployed in O'Brien Bay, approximately 5 kilometres south of Casey station, East Antarctica, in the austral summer of 2014/15. Surface and sub-surface (in water below the sea ice) infrastructure allowed controlled manipulation of seawater pH levels (reduced by 0.4 pH units below ambient) in 2 chambers placed on the sea floor over natural benthic communities. Two control chambers (no pH manipulation) and two open plots (no chambers, no pH manipulation) were also sampled to compare to the pH manipulated (acidified) treatment chambers. Details of the antFOCE experiment can be found in the report – "antFOCE 2014/15 – Experimental System, Deployment, Sampling and Analysis". This report and a diagram indicating how the various antFOCE data sets relate to each other are available at: https://data.aad.gov.au/metadata/records/AAS_4127_antFOCE_Project4127 High throughput sequencing of the 16S rRNA gene (Shane Powell) Genomic DNA samples were sequenced at the Ramaciotti Centre for Genomics at the University of New South Wales. The V4 region of the 16S rRNA gene was sequenced with the primers 515F – 806R on an Illumina MiSeq with MiSeq v2 reagent kit. Sequences were processed using MOTHUR v 1.36.1 (Schloss et al. 2009) following the suggested protocol for processing MiSeq datasets as described in Kozich et al. (2013) with the following modifications. The make.contigs command was used to join the paired-end reads from the fastaq files. Sequences that were longer than 300 bp or contained more than one ambiguous base were removed with the screen.seqs. Within each sample, exact duplicate sequences were merged with unique.seqs. The sequences were then aligned against the Silva database (downloaded March 1 2016). Sequences with 3 or less nucleotide differences in total were clustered together using pre.cluster. Potentially chimeric sequences were removed with the defaults settings of the MOTHUR implementation of uchime. After removal of chimeric sequences, the remaining sequences were grouped into operational taxonomic units (OTU) using cluster.split with taxlevel=4 (Order). Finally a table of the number of times each OTU appeared in each sample was generated with make.shared with a cut-off of 0.03 and the OTU were classified with classify.otu. As the sample with the fewest sequences contained 63955 sequences, rarefaction was carried out using the sub.sample command to randomly select 63 955 sequences per sample. A total of 4 604 760 sequences remained in the final OTU table. Any OTU that contained less than 500 sequences (less than 0.01%) were removed as potentially spurious or chimeric sequences, especially as these were generally unclassified sequences. Multivariate analyses were carried out using the PRIMER software. Data were standardised (converted to a percentage) prior to any other analysis.... Kozich, J.J., Westcott, S.L., Baxter, N.T., Highlander, S.K. and Schloss, P.D., 2013. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and environmental microbiology 79:5112-5120. Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M., Hollister, E.B., Lesniewski, R.A., Oakley, B.B., Parks, D.H., Robinson, C.J. and Sahl, J.W., 2009. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Applied and environmental microbiology 75:7537-7541.

Long-term experiment on increased CO2 level on krill physiology. Krill were exposed to a range of CO2 conditions 400-4000ppm over a year, and their growth, mortality, and physiology were monitored. -List of files- Ericson Krill Ocean Acidification Study Raw Data_for data centre.xlsx: This file contains data on krill growth, mortality, physiology, and biochemistry, as well as information on water chemistry throughout 1 year period of the experiment. Ericson et al. Adult krill OA MS final submission.pdf: Unpublished manuscript of the experiment including all methods of the experiment.

Carbonate chemistry data for the antFOCE seawater samples. The download file contains an Excel spreadsheet with a number of worksheets detailing the samples collected from O'Brien Bay, Casey Station. The dataset includes information on oxygen levels, pH levels, temperature and salinity levels, as well as the concentrations of various elements (dissolved inorganic carbon, phosphate, nitrate, nitrite, silicate). Free-ocean CO2 enrichment (FOCE) experiments have been deployed in marine ecosystems to manipulate carbonate system conditions to those predicted in future oceans. We investigated whether the pH/carbonate chemistry of extremely cold polar waters can be manipulated in an ecologically relevant way, to represent conditions under future atmospheric CO2 levels, in an in-situ FOCE experiment in Antarctica. We examined spatial and temporal variation in local ambient carbonate chemistry at hourly intervals at two sites between December and February and compared these with experimental conditions. We successfully maintained a mean pH offset in acidified benthic chambers of -0.38 (plus or minus 0.07) from ambient for approximately 8 weeks. Local diel and seasonal fluctuations in ambient pH were duplicated in the FOCE system. Large temporal variability in acidified chambers resulted from system stoppages. The mean pH, Ωarag and fCO2 values in the acidified chambers were 7.688 plus or minus 0.079, 0.62 plus or minus 0.13 and 912 plus or minus 150 micro-atm respectively. Variation in ambient pH appeared to be mainly driven by salinity and biological production and ranged from 8.019 to 8.192 with significant spatio-temporal variation. This experiment demonstrates the utility of FOCE systems to create conditions expected in future oceans that represent ecologically relevant variation, even under polar conditions.

Metadata record for data from AAS (ASAC) project 3134. Data from this project will be available via the child records. Public Ocean acidification and warming are global phenomena that will impact marine biota through the 21st century. This project will provide urgently needed predictive information on the likely survivorship of benthic invertebrates in near shore Antarctic environments that is crucial for risk assessment of potential future changes to oceans. As oceans acidify carbonate saturation decreases, reducing the material required to produce marine skeletons. By examining the effects of increased ocean temperature and acidification on planktonic and benthic life stages of both calcifying and non-calcifying ecologically important organisms, predictions can be made on the potential vulnerability of marine biota to climatic change. Project Objectives: This project aims to deliver one of the first assessments of the impacts that ocean warming and acidification through rising CO2 levels will have on Antarctic benthic marine invertebrates and of the adaptive capacity of common Antarctic biota to climate change. The developmental success of species that have a skeleton will be compared to those that do not under controlled conditions of increased sea water temperature and CO2. A comparison of the responses and sensitivity of developmental stages of calcifiers (echinoids, bivalves) and non-calcifiers (asteroids) to elevated CO2 and temperature will generate much needed empirical data for assessment of risk and adaptive capacity of Antarctica's marine biota and will enable predictions of how benthic invertebrates will fare with respect to climate change scenarios. The specific aims of the project are to: 1 - examine the impacts of predicted future elevated ocean temperatures and CO2 on fertilisation success, embryonic and larval development of Antarctic molluscs and echinoderms 2 - document skeletal calcification and morphology and growth in larvae under controlled conditions of increased sea water temperature and CO2. 3 - compare the dynamics of biomineralisation with respect to the elemental composition in response to increased temperature and CO2 in species with aragonite and calcite exoskeletons (bivalves) and porous high magnesium calcite endoskeletons (echinoids) to assess the potential for an in-built adaptive response in calcification 4 - used as a biomarker measure of stress and impaired calcification. 5 - compare biomineralisation and elemental signatures in skeletons in larvae of Antarctic molluscs and echinoderms under climate change scenarios with that determined for related species at lower latitudes to assess the relative sensitivity and vulnerability of Antarctic biota. Taken from the 2009-2010 Progress Report: Progress against objectives: 1. Unsuccessful as target species, Sterechinus neumayeri had already passed its spawning period, and attempts to spawn and fertilise the Antarctic bivalve, Laternula ellipticaskeleta failed. 2. Skeletal calcification and morphology of juveniles of Abatus nimrodi were successfully documented under controlled conditions of ocean warming and acidification. 3. Juveniles of A. nimrodi were preserved and returned to Australia in order to compare the dynamics of biomineralisation and skeletal mineralogy. 4. No heat shock protein experiments were carried out. 5. Air-dried tests of S. neumayeri and A. nimrodi were RTA'd in order to compare the dynamics of biomineralisation and skeletal mineralogy. Taken from some project abstracts written by two students working on the project: Impacts of ocean acidification and increasing seawater temperature on the early life history of the Antarctic echinoderm Sterechinus neumayeri. Simultaneous effects of ocean acidification and temperature change in Antarctic environments warrant investigation as little is known about the synergistic consequences of these parameters on Antarctic benthic species. Fertilisation success, embryo cleavage, blastulation and gastrulation were documented in the sea urchin Sterechinus neumayeri, reared for up to 12 days under experimental pCO2 and elevated temperature scenarios predicted by the IPCC (2007) over the next century. Experimental treatments included controls (-1 degrees C, pH 8.0), elevated temperature (1 degrees C, 3 degrees C) and decreased pH (7.8, 7.6) in all combinations in a multi-factorial design. Preliminary results suggest that fertilisation and development up to the gastrula stages are robust to increases in pCO2 and temperature predicted by the year 2100. Percentages of normally developing blastula and gastrula were also slightly higher in temperatures 2 degrees C above ambient. Impacts of ocean acidification and increasing seawater temperature on juveniles of two Antarctic heart urchins, Abatus ingens and Abatus shackletoni. Simultaneous effects of ocean acidification and temperature change in Antarctic environments warrant investigation as little is known about the synergistic consequences of these factors on Antarctic benthic species. Juvenile Abatus ingens and Abatus shackletoni were incubated under experimental pCO2 and elevated temperature scenarios reflective of those predicted by the IPCC (2007). Direct development from embryos to juveniles occurs in these species without a pelagic larval phase and the developing young are lecithotrophic for an extended period. Adult urchins were collected near Davis Station during the Austral summer season (January-February 2011). Juveniles were extracted from the parental brood pouch and reared in flow-through experimental treatments for 4 weeks. CO2-enriched air was supplied to seawater in which pCO2 was regulated at the target levels of 448 plus or minus 6.51 (pH 8.01 plus or minus 0.005), 846 plus or minus 6.58 (pH 7.83 plus or minus 0.005) and 1371 plus or minus 7.34 (pH 7.63 plus or minus 0.007) ppm and seawater temperature was set at -1 plus or minus 0.03 degrees C (Control) and 1 plus or minus 0.32 degrees C. Preliminary results from this investigation showed significant increases in spine growth in juveniles of both A.ingens and A. shackletoni over the experimental period. However, juveniles reared in 1 degrees C significantly exhibited more incidences of epithelial separation in the spines compared to those reared in -1 degrees C. This suggests that, although there is an inherent capacity for tolerance of varying levels of pH in seawater in the absence of the protection afforded by the maternal brood pouch, these juveniles are still at risk from increasing temperatures.

The effect of pH, temperature and sperm concentration on the fertilisation of Sterechinus neumayeri was investigated. Adult Sterechinus neumayeri were collected from Ellis Fjord Narrows between December and January 2011-12 and held in the Ecotox Field Aquarium Module until used. Between 3-4 male and female individuals were spawned using 0.5M KCl and gametes were collected separately before being fertilised in treatment. The data set shows the percentage of fertilised and non-fertilised eggs of Sterechinus neumayeri scored at 20h post-fertilisation. Eggs were fertilised in various combinations of pH, temperature and sperm concentration treatments (pH: 8.0 (Control), 7.8 and 7.6; Temperature: 1 degrees C (Control), 3 degrees C and 5 degrees C; Sperm concentration (sperm:egg ratio): 1000:1 (Control), 750:1, 250: 1, 50:1 and 5:1). At 20h post fertilisation, 5 ml aliquot was removed from fertilisation vials and eggs were counted and determined if they were fertilised or not. Seawater parameters of treatments were measured at the start and end of the experiment. Detailed information of the spreadsheets are as follows: Seawater Parameters column headings: Temperature - measured in degrees C , shows the temperature treatments used pH - shows the pH levels used Subheading pH - pH level measured for the day using NIST certified buffers Subheading MV - pH level measured for the day in millivolts Subheading Total pH - total pH level in seawater obtained from MV measurements Subheading Temp - temperature of seawater measured for the day 1 deg C column headings: Experiment - number of experiments pH - shows the pH for each treatment Sperm Concentration - shows the sperm concentration used for each treatment in a egg:sperm ratio Rep - shows the number of replicates per experiment Unfertilised eggs - eggs without visible fertilisation envelope and no cleavage after 20h Fertilised eggs - eggs with visible fertilisation envelope and/or cleavage after 20h Fertilised deformed eggs - eggs with visible fertilisation envelope but deformed Total eggs - total eggs scored (whether fertilised or unfertilised) % Fertilised - fertilised eggs (deformed and non-deformed)/Total eggs 3 deg C and 5 deg C have the same column headings as 1 deg C. AAS3134 Abatus sp Growth Experiment Davis 2011-12: The effect of pH and temperature on the growth rate of juvenile Abatus ingens and Abatus shackletoni were investigated. Adult Abatus were collected off Airport Beach in waters 4-5m depth. Data set shows the growth rate of juveniles of Abatus ingens and Abatus shackletoni after a 4-week exposure to various combinations of pH and temperature. Juveniles of each species was removed from maternal pouches and photographed on the oral side before being exposed to combinations of pH (8.0 (Control), 7.8 and 7.6) and temperature (-1 degrees C (Control) and 1 degrees C) levels. They were incubated in treatments for 4 weeks before being removed and rephotographed. The lengths of 10 spines per juvenile were measured in the pre- and post-experiment photographs using ImageJ and the difference calculated to get a growth rate per juvenile. Seawater parameters of treatments were measured at the beginning of the experiment and subsequently once a day until the end of the experiment. Detailed information of the spreadsheets are as follows: A ingens (pre-exp) i.e. juvenile Abatus ingens spine lengths measured before exposure to experimental treatments. Column headings are: Spine number and length (mm): Length of each spine (1 - 10) measured per juvenile in mm. R1 - R12: Number of juveniles A ingens (post-exp) i.e. juvenile Abatus ingens spine lengths measured after 4-week exposure to experimental treatments. Column headings are identical to the above. A shackletoni (pre-exp) i.e. juvenile Abatus shackletoni spine lengths measured before exposure to experimental treatments. Column headings are identical to the above. A shackletoni (post-exp) i.e. juvenile Abatus shackletoni spine lengths measured after 4-week exposure to experimental treatments. Column headings are identical to the above. 2011-12 Aquarium pH and temp main headings show different treatment parameters. Column sub-headings are: Date - Date of measured seawater parameters Salinity - salinity of seawater measured Ppm - Amount of CO2 gas pumped into water recorded in parts per million pH - measured pH of seawater using NIST-certified buffers MV - pH of seawater recorded in millivolts Total pH - total pH of seawater derived from MV Temp - Temperature of seawater measured in degrees C.

These data sets describe the toxicity of lead, coppyer, zinc and cadmium spiked seawater to the juveniles of Abatus ingens and Abatus nimrodi. Metals were tested separately over exposure periods of 96 and 240 hours. The experimental endpoint was mortality as defined by cessation of observable movement. The coding system for the data files is J(juvenile)_An (Abatus nimrodi) or Ai (Abatus ingens) - Metal name_Dates of the experiment_ the period of the observation (96 hour or 240 hour). This work falls under the umbrella project ASAC_2201. The fields in this dataset are: Species Toxicant Date Replicate Concentration Moving pH Salinity Dissolved Oxygen