#METABOLOMICS WORKBENCH dagaul_20210722_122433 DATATRACK_ID:2765 STUDY_ID:ST001885 ANALYSIS_ID:AN003049 PROJECT_ID:000000 VERSION 1 CREATED_ON July 22, 2021, 1:20 pm #PROJECT PR:PROJECT_TITLE Differentiating toxic and nontoxic cogeneric harmful algae using the non-polar PR:PROJECT_TITLE metabolome. PR:PROJECT_SUMMARY Recognition and rejection of chemically defended prey is critical to maximizing PR:PROJECT_SUMMARY fitness for predators. Paralytic shellfish toxins (PSTs) which strongly inhibit PR:PROJECT_SUMMARY voltage-gated sodium channels in diverse animal taxa are produced by several PR:PROJECT_SUMMARY species of the bloom-forming algal genus Alexandrium where they appear to PR:PROJECT_SUMMARY function as chemical defenses against grazing copepods. Despite PSTs being PR:PROJECT_SUMMARY produced and localized within phytoplankton cells, some copepods distinguish PR:PROJECT_SUMMARY toxic from non-toxic prey, selectively ingesting less toxic cells, in ways that PR:PROJECT_SUMMARY suggest cell surface recognition perhaps associated with non-polar metabolites. PR:PROJECT_SUMMARY In this study LC/MS and NMR-based metabolomics revealed that the non-polar PR:PROJECT_SUMMARY metabolomes of two toxic species (Alexandrium catenella and Alexandrium PR:PROJECT_SUMMARY pacificum) vary considerably from their non-toxic congener Alexandrium tamarense PR:PROJECT_SUMMARY despite all three being very closely related. Toxic and non-toxic Alexandrium PR:PROJECT_SUMMARY spp. were distinguished from each other by metabolites belonging to seven lipid PR:PROJECT_SUMMARY classes. Of these, 17 specific metabolites were significantly more abundant in PR:PROJECT_SUMMARY both toxic A. catenella and A. pacificum compared to non-toxic A. tamarense PR:PROJECT_SUMMARY suggesting that just a small portion of the observed metabolic variability is PR:PROJECT_SUMMARY associated with toxicity. Future experiments aimed at deciphering chemoreception PR:PROJECT_SUMMARY mechanisms of copepod perception of Alexandrium toxicity should consider these PR:PROJECT_SUMMARY metabolites, and the broader lipid classes phosphatidylcholines and sterols, as PR:PROJECT_SUMMARY potential candidate cues. PR:INSTITUTE Georgia Institute of Technology PR:LAST_NAME Brown PR:FIRST_NAME Emily PR:ADDRESS 950 Atlantic Dr Atlanta GA 30332, USA PR:EMAIL julia.kubanek@biosci.gatech.edu PR:PHONE 404-894-8424 #STUDY ST:STUDY_TITLE MS Differentiating toxic and nontoxic cogeneric harmful algae using the ST:STUDY_TITLE non-polar metabolome. ST:STUDY_SUMMARY Recognition and rejection of chemically defended prey is critical to maximizing ST:STUDY_SUMMARY fitness for predators. Paralytic shellfish toxins (PSTs) which strongly inhibit ST:STUDY_SUMMARY voltage-gated sodium channels in diverse animal taxa are produced by several ST:STUDY_SUMMARY species of the bloom-forming algal genus Alexandrium where they appear to ST:STUDY_SUMMARY function as chemical defenses against grazing copepods. Despite PSTs being ST:STUDY_SUMMARY produced and localized within phytoplankton cells, some copepods distinguish ST:STUDY_SUMMARY toxic from non-toxic prey, selectively ingesting less toxic cells, in ways that ST:STUDY_SUMMARY suggest cell surface recognition perhaps associated with non-polar metabolites. ST:STUDY_SUMMARY In this study LC/MS and NMR-based metabolomics revealed that the non-polar ST:STUDY_SUMMARY metabolomes of two toxic species (Alexandrium catenella and Alexandrium ST:STUDY_SUMMARY pacificum) vary considerably from their non-toxic congener Alexandrium tamarense ST:STUDY_SUMMARY despite all three being very closely related. Toxic and non-toxic Alexandrium ST:STUDY_SUMMARY spp. were distinguished from each other by metabolites belonging to seven lipid ST:STUDY_SUMMARY classes. Of these, 17 specific metabolites were significantly more abundant in ST:STUDY_SUMMARY both toxic A. catenella and A. pacificum compared to non-toxic A. tamarense ST:STUDY_SUMMARY suggesting that just a small portion of the observed metabolic variability is ST:STUDY_SUMMARY associated with toxicity. Future experiments aimed at deciphering chemoreception ST:STUDY_SUMMARY mechanisms of copepod perception of Alexandrium toxicity should consider these ST:STUDY_SUMMARY metabolites, and the broader lipid classes phosphatidylcholines and sterols, as ST:STUDY_SUMMARY potential candidate cues. ST:INSTITUTE Georgia Institute of Technology ST:LAST_NAME Brown ST:FIRST_NAME Emily ST:ADDRESS 950 Atlantic Dr Atlanta GA 30332, USA ST:EMAIL julia.kubanek@biosci.gatech.edu ST:PHONE 404-894-8424 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Alexandrium catenella; Alexandrium tamarense; Alexandrium pacificum SU:SPECIES_GROUP Not Human -- Alexandrium catenella; Alexandrium tamarense; Alexandrium pacificum #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_1 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_1 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_2 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_2 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_3 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_3 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_4 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_4 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_5 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_5 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_6 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_6 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_7 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_7 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_8 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_8 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_9 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_9 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_10 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_10 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_11 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_11 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_12 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_12 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_13 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_13 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AC_14 factor:Treatment;Alexandrium_catenella RAW_FILE_NAME=74_3ERB74_AC_14 SUBJECT_SAMPLE_FACTORS - 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74_3ERB74_AT_10 factor:Treatment;Alexandrium_tamarense RAW_FILE_NAME=74_3ERB74_AT_10 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AT_11 factor:Treatment;Alexandrium_tamarense RAW_FILE_NAME=74_3ERB74_AT_11 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AT_12 factor:Treatment;Alexandrium_tamarense RAW_FILE_NAME=74_3ERB74_AT_12 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AT_13 factor:Treatment;Alexandrium_tamarense RAW_FILE_NAME=74_3ERB74_AT_13 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AT_14 factor:Treatment;Alexandrium_tamarense RAW_FILE_NAME=74_3ERB74_AT_14 SUBJECT_SAMPLE_FACTORS - 74_3ERB74_AT_15 factor:Treatment;Alexandrium_tamarense RAW_FILE_NAME=74_3ERB74_AT_15 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AP_1 factor:Treatment;Alexandrium_pacificum RAW_FILE_NAME=75_3ERB75_AP_1 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AP_2 factor:Treatment;Alexandrium_pacificum RAW_FILE_NAME=75_3ERB75_AP_2 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AP_3 factor:Treatment;Alexandrium_pacificum RAW_FILE_NAME=75_3ERB75_AP_3 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AP_4 factor:Treatment;Alexandrium_pacificum RAW_FILE_NAME=75_3ERB75_AP_4 SUBJECT_SAMPLE_FACTORS - 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SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_5 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_5 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_6 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_6 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_7 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_7 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_8 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_8 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_9 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_9 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_10 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_10 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_11 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_11 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_12 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_12 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_13 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_13 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_14 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_14 SUBJECT_SAMPLE_FACTORS - 75_3ERB75_AT_15 factor:Treatment;Alexandrium_tamarense2 RAW_FILE_NAME=75_3ERB75_AT_15 SUBJECT_SAMPLE_FACTORS - Blank01 factor:QC RAW_FILE_NAME=Blank01 SUBJECT_SAMPLE_FACTORS - Blank02 factor:QC RAW_FILE_NAME=Blank02 SUBJECT_SAMPLE_FACTORS - QC01 factor:QC RAW_FILE_NAME=QC01 SUBJECT_SAMPLE_FACTORS - QC02 factor:QC RAW_FILE_NAME=QC02 SUBJECT_SAMPLE_FACTORS - QC03 factor:QC RAW_FILE_NAME=QC03 SUBJECT_SAMPLE_FACTORS - QC04 factor:QC RAW_FILE_NAME=QC04 SUBJECT_SAMPLE_FACTORS - QC05 factor:QC RAW_FILE_NAME=QC05 SUBJECT_SAMPLE_FACTORS - QC06 factor:QC RAW_FILE_NAME=QC06 SUBJECT_SAMPLE_FACTORS - QC07 factor:QC RAW_FILE_NAME=QC07 SUBJECT_SAMPLE_FACTORS - QC08 factor:QC RAW_FILE_NAME=QC08 SUBJECT_SAMPLE_FACTORS - QC09 factor:QC RAW_FILE_NAME=QC09 SUBJECT_SAMPLE_FACTORS - QC10 factor:QC RAW_FILE_NAME=QC10 #COLLECTION CO:COLLECTION_SUMMARY Alexandrium cells were collected by vacuum filtration onto GF/F filters and CO:COLLECTION_SUMMARY quenched with liquid nitrogen. Frozen cells with filters were stored in CO:COLLECTION_SUMMARY foil(previously muffled for 3 h at 450 °C) at -80 °C until extraction. CO:SAMPLE_TYPE Algae #TREATMENT TR:TREATMENT_SUMMARY Metabolomes of toxic versus non-toxic species were compared using the following TR:TREATMENT_SUMMARY experimental pairings: A. tamarense (n=15) with A. catenella (n=15) (Experiment TR:TREATMENT_SUMMARY 1) and A. tamarense (n=15) with A. pacificum (n=15) (Experiment 2). The same TR:TREATMENT_SUMMARY non-toxic strain of A. tamarense was used in both experiments but the two TR:TREATMENT_SUMMARY experiments were conducted separately, in different months, to make the TR:TREATMENT_SUMMARY experiment manageable based on availability of batches grown from stock TR:TREATMENT_SUMMARY cultures. For both experiments, Alexandrium spp. cultures at a cell density of TR:TREATMENT_SUMMARY 12,000 to 13,000 cells mL-1 were split into fifteen 300 mL subcultures of each TR:TREATMENT_SUMMARY species which grew for two days. At the end of each experiment, during TR:TREATMENT_SUMMARY harvesting, a 1.0 mL aliquot from each culture was preserved with Lugol’s TR:TREATMENT_SUMMARY solution to measure cell concentrations #SAMPLEPREP SP:SAMPLEPREP_SUMMARY IPA: ACN (2:1) extract 4.54e6 cells per 1mL #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Accucore C30 (150 x 2.1 mm, 2.6um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap ID-X tribrid MS:INSTRUMENT_TYPE orbitrap and ion trap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS compound discoverer MS:MS_RESULTS_FILE ST001885_AN003049_Results.txt UNITS:peak area Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END