#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
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
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
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	
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:SAMPLE_TYPE                   	Cell extract
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.
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.
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: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
AN:ANALYSIS_TYPE                 	MS
MS:MS_COMMENTS                   	-
MS:INSTRUMENT_NAME               	Waters Synapt G2 QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:CAPILLARY_VOLTAGE             	2.0
MS:MS_RESULTS_FILE               	ST000921_AN001510_Results.txt	UNITS:Normalized Abundance