#METABOLOMICS WORKBENCH srjhogan@gatech.edu_20180119_081950 DATATRACK_ID:1302 STUDY_ID:ST000921 ANALYSIS_ID:AN001510 PROJECT_ID:PR000637 VERSION 1 CREATED_ON January 19, 2018, 1:56 pm #PROJECT PR:PROJECT_TITLE Karenia brevis allelopathy compromises the lipidome, membrane integrity, and PR:PROJECT_TITLE photosynthetic efficiency of competitors PR:PROJECT_TYPE Untargeted Lipidomics PR:PROJECT_SUMMARY Comparing effects on lipidome of phytoplankton competitors based on exposure to PR:PROJECT_SUMMARY K. Brevis PR:INSTITUTE Georgia Institute of Technology PR:DEPARTMENT Chemistry PR:LABORATORY Fernández PR:LAST_NAME Hogan PR:FIRST_NAME Scott PR:ADDRESS 901 Atlantic Drive, Atlanta, GA, 30332, USA PR:EMAIL srjhogan@gatech.edu PR:PHONE 2156924657 #STUDY ST:STUDY_TITLE Karenia brevis allelopathy compromises the lipidome, membrane integrity, and ST:STUDY_TITLE photosynthetic efficiency of competitors ST:STUDY_TYPE Untargeted lipidomics ST:STUDY_SUMMARY Allelopathy, or the release of compounds that inhibit competitors, is a form of ST:STUDY_SUMMARY interference competition that is common among bloom-forming phytoplankton. ST:STUDY_SUMMARY Allelopathy is hypothesized to play a role in bloom propagation and maintenance ST:STUDY_SUMMARY and is well established in the red tide dinoflagellate Karenia brevis. K. brevis ST:STUDY_SUMMARY typically suppresses competitor growth through unknown mechanisms over the ST:STUDY_SUMMARY course of many days. When we investigated the effects of allelopathy on the ST:STUDY_SUMMARY lipidomes of two competing phytoplankton, Asterionellopsis glacialis and ST:STUDY_SUMMARY Thalassiosira pseudonana using nuclear magnetic resonance (NMR) spectroscopy and ST:STUDY_SUMMARY mass spectrometry (MS)- based metabolomics, we found that the lipidomes of both ST:STUDY_SUMMARY species were significantly altered, however A. glacialis maintained a more ST:STUDY_SUMMARY robust response whereas T. pseudonana saw significant alterations in fatty acid ST:STUDY_SUMMARY synthesis, cell membrane integrity, and a decrease in photosynthetic efficiency. ST:STUDY_SUMMARY Membrane- associated lipids were significantly suppressed for T. pseudonana ST:STUDY_SUMMARY exposed to allelopathy to the point of permeabilizing the cell membrane of ST:STUDY_SUMMARY living cells. The dominant mechanisms of K. brevis allelopathy appear to target ST:STUDY_SUMMARY lipid biosynthesis affecting multiple physiological pathways suggesting that ST:STUDY_SUMMARY exuded compounds have the ability to significantly alter competitor physiology ST:STUDY_SUMMARY and give K. brevis a competitive edge over sensitive species. ST:INSTITUTE Georgia Institute of Technology ST:DEPARTMENT Chemistry ST:LABORATORY Fernández ST:LAST_NAME Hogan ST:FIRST_NAME Scott ST:ADDRESS 901 Atlantic Drive, Atlanta, GA, 30332, USA ST:EMAIL srjhogan@gatech.edu ST:PHONE 2156924657 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 51 #SUBJECT SU:SUBJECT_TYPE Plankton SU:SUBJECT_SPECIES Thalassiosira pseudonana;Karenia brevis #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - AgC2 Class:Control SUBJECT_SAMPLE_FACTORS - AgC10 Class:Control SUBJECT_SAMPLE_FACTORS - AgC11 Class:Control SUBJECT_SAMPLE_FACTORS - AgC6 Class:Control SUBJECT_SAMPLE_FACTORS - AgC1 Class:Control SUBJECT_SAMPLE_FACTORS - AgC3 Class:Control SUBJECT_SAMPLE_FACTORS - AgC5 Class:Control SUBJECT_SAMPLE_FACTORS - AgC4 Class:Control SUBJECT_SAMPLE_FACTORS - AgC8 Class:Control SUBJECT_SAMPLE_FACTORS - AgC15 Class:Control SUBJECT_SAMPLE_FACTORS - AgC9 Class:Control SUBJECT_SAMPLE_FACTORS - AgC7 Class:Control SUBJECT_SAMPLE_FACTORS - AgC12 Class:Control SUBJECT_SAMPLE_FACTORS - AgT2 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT15 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT8 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT7 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT10 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT9 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT5 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT12 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT6 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT4 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT3 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT11 Class:treatment SUBJECT_SAMPLE_FACTORS - AgT1 Class:treatment SUBJECT_SAMPLE_FACTORS - TpC2 Class:Control SUBJECT_SAMPLE_FACTORS - TpC7 Class:Control SUBJECT_SAMPLE_FACTORS - TpC5 Class:Control SUBJECT_SAMPLE_FACTORS - TpC10 Class:Control SUBJECT_SAMPLE_FACTORS - TpC8 Class:Control SUBJECT_SAMPLE_FACTORS - TpC3 Class:Control SUBJECT_SAMPLE_FACTORS - TpC15 Class:Control SUBJECT_SAMPLE_FACTORS - TpC11 Class:Control SUBJECT_SAMPLE_FACTORS - TpC6 Class:Control SUBJECT_SAMPLE_FACTORS - TpC4 Class:Control SUBJECT_SAMPLE_FACTORS - TpC12 Class:Control SUBJECT_SAMPLE_FACTORS - TpC9 Class:Control SUBJECT_SAMPLE_FACTORS - TpC13 Class:Control SUBJECT_SAMPLE_FACTORS - TpT8 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT5 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT13 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT10 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT2 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT9 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT4 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT7 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT3 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT6 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT15 Class:treatment SUBJECT_SAMPLE_FACTORS - TpT11 Class:treatment #COLLECTION CO:COLLECTION_SUMMARY Briefly, diatoms Thalassiosira pseudonana strain CCMP 1335 and Asterionellopsis CO:COLLECTION_SUMMARY glacialis strain CCMP 137 were grown in silicate-amended L1 media in artificial CO:COLLECTION_SUMMARY seawater (Instant Ocean, 35 ppt). Karenia brevis strain CCMP 2228 was cultured CO:COLLECTION_SUMMARY in similar conditions above with L1 media-amended artificial seawater. All CO:COLLECTION_SUMMARY cultures were maintained at 21 ˚C with a 12:12 light/dark cycle and an CO:COLLECTION_SUMMARY irradiance of 100-145 µmol/m2s in a Percival incubator (Biospherical Instrument CO:COLLECTION_SUMMARY QSL2100). CO:SAMPLE_TYPE Cell extract #TREATMENT TR:TREATMENT_SUMMARY To expose diatoms to competition with allelopathic K. brevis, K. brevis was TR:TREATMENT_SUMMARY co-cultured with each of the two diatom species (n=14 per species). K. brevis TR:TREATMENT_SUMMARY was grown inside a permeable dialysis membrane to allow for exchange of exuded TR:TREATMENT_SUMMARY allelopathic compounds without direct interaction of K. brevis and diatom cells, TR:TREATMENT_SUMMARY which were grown in flasks in which the dialysis tubes were placed. Control TR:TREATMENT_SUMMARY cultures consisted of dialysis membranes (molecular weight cutoff, 50 kDa) TR:TREATMENT_SUMMARY filled with L1 media diluted to conditions similar to that of exponential growth TR:TREATMENT_SUMMARY phase K. brevis (n = 15 per diatom species) in place of diatom species. This TR:TREATMENT_SUMMARY co-culture experiment was halted once competitor cultures reached exponential TR:TREATMENT_SUMMARY growth stage, which was 6 d for T. pseudonana and 8 d for A. glacialis, after TR:TREATMENT_SUMMARY which diatom cells were filtered onto GF/C filters (Whatman #1922-110, muffled TR:TREATMENT_SUMMARY at 450 ˚C for 3 h) and dipped into liquid nitrogen to quench intracellular TR:TREATMENT_SUMMARY metabolism. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To separate polar and lipid intracellular metabolites, dried extracts were SP:SAMPLEPREP_SUMMARY dissolved in a biphasic mixture of 9:10:15 water/methanol/chloroform. The more SP:SAMPLEPREP_SUMMARY lipophilic layer was removed and washed twice with 9:10 water/methanol. Lipid SP:SAMPLEPREP_SUMMARY extracts were reconstituted in 200 μL 2-propanol. Quantitative metabolomics SP:SAMPLEPREP_SUMMARY data were acquired using a Waters Xevo G2 QTOF mass spectrometer. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase MS:INSTRUMENT_NAME Waters Synapt G2 QTOF CH:COLUMN_NAME Waters Acquity BEH C18 (50 x 2.1mm, 1.7um) CH:FLOW_GRADIENT 0-1 min, 70% B; 1-3 min, 75% B; 3-6 min, 80% B; 6-10 min, 90% B; 10-14 min, 100% CH:FLOW_GRADIENT B. CH:FLOW_RATE .3 mL/min CH:COLUMN_TEMPERATURE 60 CH:SOLVENT_A water: acetonitrile (40:60) +10 mM ammonium formate + 0.1% formic acid CH:SOLVENT_B 10% acetonitrile in 2-propanol +10 mM ammonium formate + 0.1% formic acid #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Waters Synapt G2 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:CAPILLARY_VOLTAGE 2.0 MS:SOURCE_TEMPERATURE 90°C MS:DESOLVATION_GAS_FLOW 600 L/h MS:DESOLVATION_TEMPERATURE 250 °C MS:MS_RESULTS_FILE ST000921_AN001510_Results.txt UNITS:Normalized Abundance #END