#METABOLOMICS WORKBENCH JKubanek_20210720_081653_mwtab.txt DATATRACK_ID:2759 STUDY_ID:ST001895 ANALYSIS_ID:AN003076 PROJECT_ID:000000 VERSION 1 CREATED_ON July 23, 2021, 2:13 pm #PROJECT PR:PROJECT_TITLE Differentiating toxic and nontoxic congeneric harmful algae using the non-polar PR:PROJECT_TITLE metabolome PR:PROJECT_TYPE Using metabolomics to compare species 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:DEPARTMENT School of Biological Sciences, School of Chemistry and Biochemistry, Center for PR:DEPARTMENT Microbial Dynamics and Infection, Parker H. Petit Institute for Bioengineering PR:DEPARTMENT and Bioscience PR:LABORATORY Kubanek Lab 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 PR:PROJECT_COMMENTS This study has 3 parts: 2 NMR (Experiment 1: A. catenella-A. tamarense & PR:PROJECT_COMMENTS Experiment 2: A. pacificum-A. tamarense) and MS PR:CONTRIBUTORS Emily R. Brown, Sam G. Moore, David A. Gaul, and Julia Kubanek #STUDY ST:STUDY_TITLE NMR Differentiating toxic and nontoxic congeneric harmful algae using the ST:STUDY_TITLE non-polar metabolome (Experiment 1) ST:STUDY_TYPE 1H NMR Metabolomics to compare toxic and non-toxic species ST:STUDY_SUMMARY Metabolomics comparison of toxic and non-toxic species of phytoplankton from the ST:STUDY_SUMMARY genus Alexandrium.This study was carried out using 2 pairwise experiments, A. ST:STUDY_SUMMARY catenella compared to A. tamarense (Experiment 1) and A. pacificum compared to ST:STUDY_SUMMARY A. tamarense (Experiment 2). This study includes the NMR data from Experiment 1. ST:INSTITUTE Georgia Institute of Technology ST:DEPARTMENT School of Biological Sciences, School of Chemistry and Biochemistry, Center for ST:DEPARTMENT Microbial Dynamics and Infection, Parker H. Petit Institute for Bioengineering ST:DEPARTMENT and Bioscience ST:LABORATORY Kubanek Lab ST:LAST_NAME Brown ST:FIRST_NAME Emily ST:ADDRESS 950 Atlantic Dr Atlanta, Georgia USA 30332 ST:EMAIL julia.kubanek@biosci.gatech.edu ST:PHONE 404-894-8424 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 30 ST:STUDY_COMMENTS Part 1 of 3. This part includes NMR analysis of non-polar metabolites from ST:STUDY_COMMENTS Experiment 1 using oPLSDA. Parts 2 and 3 inlcude NMR analysis of non-polar ST:STUDY_COMMENTS metabolites from Experiment 2 and the corresponding mass spectrometry ST:STUDY_COMMENTS metabolomics for both Experiments 1 and 2. #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Alexandrium catenella; Alexandrium tamarense; Alexandrium pacificum SU:TAXONOMY_ID 2925; 2926 SU:GENOTYPE_STRAIN CCMP 1719; CCMP 2023 SU:GENDER Not applicable #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 - Acatenella 1 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 1; Cells_per_mL=15596; Number_of_cells_extracted=3883404; NMR_solvent_volume (uL)=240; MS_solvent_volume (uL)=855 SUBJECT_SAMPLE_FACTORS - Acatenella 2 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 2; Cells_per_mL=16338; Number_of_cells_extracted=4068162; NMR_solvent_volume (uL)=251; MS_solvent_volume (uL)=895 SUBJECT_SAMPLE_FACTORS - Acatenella 3 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 3; Cells_per_mL=14482; Number_of_cells_extracted=3606018; NMR_solvent_volume (uL)=223; MS_solvent_volume (uL)=794 SUBJECT_SAMPLE_FACTORS - Acatenella 4 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 4; Cells_per_mL=13491; Number_of_cells_extracted=3359259; NMR_solvent_volume (uL)=208; MS_solvent_volume (uL)=739 SUBJECT_SAMPLE_FACTORS - Acatenella 5 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 5; Cells_per_mL=15101; Number_of_cells_extracted=3760149; NMR_solvent_volume (uL)=232; MS_solvent_volume (uL)=828 SUBJECT_SAMPLE_FACTORS - Acatenella 6 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 6; Cells_per_mL=15101; Number_of_cells_extracted=3760149; NMR_solvent_volume (uL)=232; MS_solvent_volume (uL)=828 SUBJECT_SAMPLE_FACTORS - Acatenella 7 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 7; Cells_per_mL=14977; Number_of_cells_extracted=3729273; NMR_solvent_volume (uL)=230; MS_solvent_volume (uL)=821 SUBJECT_SAMPLE_FACTORS - Acatenella 8 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 8; Cells_per_mL=13863; Number_of_cells_extracted=3451887; NMR_solvent_volume (uL)=213; MS_solvent_volume (uL)=760 SUBJECT_SAMPLE_FACTORS - Acatenella 9 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 9; Cells_per_mL=12996; Number_of_cells_extracted=3236004; NMR_solvent_volume (uL)=200; MS_solvent_volume (uL)=712 SUBJECT_SAMPLE_FACTORS - Acatenella 10 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 10; Cells_per_mL=13120; Number_of_cells_extracted=3266880; NMR_solvent_volume (uL)=202; MS_solvent_volume (uL)=719 SUBJECT_SAMPLE_FACTORS - Acatenella 11 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 11; Cells_per_mL=13491; Number_of_cells_extracted=3359259; NMR_solvent_volume (uL)=208; MS_solvent_volume (uL)=739 SUBJECT_SAMPLE_FACTORS - Acatenella 12 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 12; Cells_per_mL=13244; Number_of_cells_extracted=3297756; NMR_solvent_volume (uL)=204; MS_solvent_volume (uL)=726 SUBJECT_SAMPLE_FACTORS - Acatenella 13 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 13; Cells_per_mL=13615; Number_of_cells_extracted=3390135; NMR_solvent_volume (uL)=210; MS_solvent_volume (uL)=746 SUBJECT_SAMPLE_FACTORS - Acatenella 14 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 14; Cells_per_mL=14358; Number_of_cells_extracted=3575142; NMR_solvent_volume (uL)=221; MS_solvent_volume (uL)=787 SUBJECT_SAMPLE_FACTORS - Acatenella 15 Treatment:Alexandrium_catenella RAW_FILE_NAME=Acatenella 15; Cells_per_mL=13120; Number_of_cells_extracted=3266880; NMR_solvent_volume (uL)=202; MS_solvent_volume (uL)=719 SUBJECT_SAMPLE_FACTORS - Atamarense 1 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 1; Cells_per_mL=16214; Number_of_cells_extracted=4037286; NMR_solvent_volume (uL)=250; MS_solvent_volume (uL)=889 SUBJECT_SAMPLE_FACTORS - Atamarense 2 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 2; Cells_per_mL=15224; Number_of_cells_extracted=3790776; NMR_solvent_volume (uL)=234; MS_solvent_volume (uL)=834 SUBJECT_SAMPLE_FACTORS - Atamarense 3 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 3; Cells_per_mL=15348; Number_of_cells_extracted=3821652; NMR_solvent_volume (uL)=236; MS_solvent_volume (uL)=841 SUBJECT_SAMPLE_FACTORS - Atamarense 4 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 4; Cells_per_mL=15224; Number_of_cells_extracted=3790776; NMR_solvent_volume (uL)=234; MS_solvent_volume (uL)=834 SUBJECT_SAMPLE_FACTORS - Atamarense 5 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 5; Cells_per_mL=15348; Number_of_cells_extracted=3821652; NMR_solvent_volume (uL)=236; MS_solvent_volume (uL)=841 SUBJECT_SAMPLE_FACTORS - Atamarense 6 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 6; Cells_per_mL=16586; Number_of_cells_extracted=4129914; NMR_solvent_volume (uL)=255; MS_solvent_volume (uL)=909 SUBJECT_SAMPLE_FACTORS - Atamarense 7 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 7; Cells_per_mL=14358; Number_of_cells_extracted=3575142; NMR_solvent_volume (uL)=221; MS_solvent_volume (uL)=787 SUBJECT_SAMPLE_FACTORS - Atamarense 8 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 8; Cells_per_mL=16214; Number_of_cells_extracted=4037286; NMR_solvent_volume (uL)=250; MS_solvent_volume (uL)=889 SUBJECT_SAMPLE_FACTORS - Atamarense 9 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 9; Cells_per_mL=14977; Number_of_cells_extracted=3729273; NMR_solvent_volume (uL)=230; MS_solvent_volume (uL)=821 SUBJECT_SAMPLE_FACTORS - Atamarense 10 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 10; Cells_per_mL=15843; Number_of_cells_extracted=3944907; NMR_solvent_volume (uL)=244; MS_solvent_volume (uL)=868 SUBJECT_SAMPLE_FACTORS - Atamarense 11 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 11; Cells_per_mL=15596; Number_of_cells_extracted=3883404; NMR_solvent_volume (uL)=240; MS_solvent_volume (uL)=855 SUBJECT_SAMPLE_FACTORS - Atamarense 12 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 12; Cells_per_mL=14729; Number_of_cells_extracted=3667521; NMR_solvent_volume (uL)=227; MS_solvent_volume (uL)=807 SUBJECT_SAMPLE_FACTORS - Atamarense 13 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 13; Cells_per_mL=13987; Number_of_cells_extracted=3482763; NMR_solvent_volume (uL)=215; MS_solvent_volume (uL)=767 SUBJECT_SAMPLE_FACTORS - Atamarense 14 Treatment:Alexandrium_tamarense RAW_FILE_NAME=Atamarense 14; Cells_per_mL=15843; Number_of_cells_extracted=3944907; NMR_solvent_volume (uL)=244; MS_solvent_volume (uL)=868 SUBJECT_SAMPLE_FACTORS - Atamarense 15 Treatment:Alexandrium_tamarense RAW_FILE_NAME=none; Cells_per_mL=15843; Number_of_cells_extracted=3944907; NMR_solvent_volume (uL)=244; MS_solvent_volume (uL)=868 #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 foil CO:COLLECTION_SUMMARY (previously muffled for 3 h at 450 °C) at -80 °C until extraction. CO:COLLECTION_PROTOCOL_FILENAME Differentiating toxic and nontoxic congeneric harmful algae using the non-polar CO:COLLECTION_PROTOCOL_FILENAME metabolome_protocol.pdf CO:SAMPLE_TYPE Algae CO:COLLECTION_METHOD Filtration and Freeze CO:STORAGE_CONDITIONS -80℃ #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 TR:TREATMENT_PROTOCOL_FILENAME Differentiating toxic and nontoxic congeneric harmful algae using the non-polar TR:TREATMENT_PROTOCOL_FILENAME metabolome_protocol.pdf TR:TREATMENT Species comparison #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Non polar extracts were dissolved with volumes calculated to account for SP:SAMPLEPREP_SUMMARY different cell densities at the time of harvesting, to a concentration SP:SAMPLEPREP_SUMMARY equivalent to 1.62 x 10^7 Alexandrium cells mL-1 in dimethyl sulfoxide d6 (99.9% SP:SAMPLEPREP_SUMMARY atom d6-DMSO; Cambridge Isotope Labs) containing 0.1% trimethylsilane (TMS) as SP:SAMPLEPREP_SUMMARY internal standard for 1H nuclear magnetic resonance (NMR) spectroscopy analysis. SP:SAMPLEPREP_SUMMARY The extracts were transferred to 3 mm NMR tubes. Extracts from Experiment 1 (A. SP:SAMPLEPREP_SUMMARY tamarense-A. catenella) were analyzed using a Bruker Avance IIIHD 800 MHz NMR SP:SAMPLEPREP_SUMMARY spectrometer equipped with a 3 mm triple resonance broadband cryoprobe. Spectra SP:SAMPLEPREP_SUMMARY of non-polar extracts from both experiments were acquired using a Bruker zg30 SP:SAMPLEPREP_SUMMARY pulse sequence 1H NMR experiment compiled from 320 scans. Due to poor NMR SP:SAMPLEPREP_SUMMARY spectral quality one A. tamarense replicate spectrum was removed from analysis SP:SAMPLEPREP_SUMMARY of Experiment 1 (A. tamarense-A. catenella. SP:SAMPLEPREP_PROTOCOL_FILENAME Differentiating toxic and nontoxic congeneric harmful algae using the non-polar SP:SAMPLEPREP_PROTOCOL_FILENAME metabolome_protocol.pdf SP:EXTRACT_STORAGE -80℃ #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker Avance IIIHD NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 800 MHz NM:NMR_PROBE 3 mm triple resonance broadband cryoprobe NM:NMR_SOLVENT Dimethyl sulfoxide d6 (99.9% atom d6-DMSO; Cambridge Isotope Labs) containing NM:NMR_SOLVENT 0.1% trimethylsilane NM:NMR_TUBE_SIZE 3 mm NM:PULSE_SEQUENCE zg30 NM:NUMBER_OF_SCANS 320 NM:BASELINE_CORRECTION_METHOD Spline NM:BINNED_INCREMENT 0.005 ppm NM:NMR_RESULTS_FILE Preprocessed_binned_Experiment1_AcvAt_NMRmetabolomics.txt UNITS:area under the curve #END