#METABOLOMICS WORKBENCH jvaorodrigues1980_20240819_081901 DATATRACK_ID:5120 STUDY_ID:ST003473 ANALYSIS_ID:AN005708 PROJECT_ID:PR002132 VERSION 1 CREATED_ON September 9, 2024, 7:25 am #PROJECT PR:PROJECT_TITLE 1H NMR metabolomics applied to assess the direct and transgenerational effects PR:PROJECT_TITLE of simvastatin on the metabolism of the amphipod Gammarus locusta PR:PROJECT_TYPE 1H NMR metabolomics to study the metabolic effects of direct exposure to PR:PROJECT_TYPE simvastatin and transgenerational effects on the polar metabolome of amphipod PR:PROJECT_TYPE Gammarus locusta PR:PROJECT_SUMMARY Pharmaceutical compounds (PhACs) and their metabolites are considered PR:PROJECT_SUMMARY contaminants of emerging concern (CECs) to public and environmental health, due PR:PROJECT_SUMMARY to their high stability, bioactivity, and persistence in conventional wastewater PR:PROJECT_SUMMARY treatment plants. Aquatic organisms can be chronically exposed to PhACs during PR:PROJECT_SUMMARY critical periods of their life or even through multi-generations. This may PR:PROJECT_SUMMARY result in severe physiological/metabolic/endocrine disturbances, not only PR:PROJECT_SUMMARY through direct exposure but also through inter- and transgenerational PR:PROJECT_SUMMARY inheritance (in the absence of the insult). Special attention is given to PR:PROJECT_SUMMARY simvastatin, one of the PhACs most prescribed to humans for the primary PR:PROJECT_SUMMARY treatment of hypercholesterolemia, known to affect endocrine functions and PR:PROJECT_SUMMARY disrupt reproduction, development, neuronal processes and/or other important PR:PROJECT_SUMMARY physiological responses; not only in exposed individuals, but also in subsequent PR:PROJECT_SUMMARY non-exposed generations. Previous studies observed that direct exposure and PR:PROJECT_SUMMARY transgenerational exposure to simvastatin has been found to impact severely on PR:PROJECT_SUMMARY crustaceans’ reproduction, growth and development, although the underlying PR:PROJECT_SUMMARY mechanism remains partially unclear. NMR metabolomic approaches may provide PR:PROJECT_SUMMARY crucial complementary mechanistic information about the cascade of metabolic PR:PROJECT_SUMMARY events occurring with exposure. In this study a comprehensive untargetd 1H NMR PR:PROJECT_SUMMARY metabolomics study was applied to measure the metabolic effects of direct PR:PROJECT_SUMMARY exposure to environmentally relevant concentrations of simvastatin (F0) and PR:PROJECT_SUMMARY transgenerational exposure (F3, where only the F0 generation was exposed) on the PR:PROJECT_SUMMARY keystone marine amphipod species Gammarus locusta. Furthermore, NMR is here PR:PROJECT_SUMMARY employed for the first time to address G. locusta metabolic behavior. The PR:PROJECT_SUMMARY obtained data added important knowledge, paving the way to an improved PR:PROJECT_SUMMARY understanding of the metabolic events cascade associated with simvastatin PR:PROJECT_SUMMARY exposure. PR:INSTITUTE University of Aveiro PR:DEPARTMENT CICECO – Aveiro Institute of Materials, Department of Chemistry PR:LABORATORY Metabolomics Group PR:LAST_NAME Rodrigues PR:FIRST_NAME Joao A. PR:ADDRESS University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, PR:ADDRESS Portugal PR:EMAIL joao.rodrigues@ua.pt PR:PHONE 00351234370707 PR:FUNDING_SOURCE This work was developed within TRANSEPIC − Exploring Transgenerational PR:FUNDING_SOURCE Epigenetic Inheritance: New Methods and Strategies to Improve Environmental PR:FUNDING_SOURCE Hazard and Risk Assessment of Key Contaminants of Emerging Concern (CECs) PR:FUNDING_SOURCE [Reference: 2022.02922.PTDC, doi: 10.54499/2022.02922.PTDC], financed by the PR:FUNDING_SOURCE Portuguese Foundation for Science and Technology (FCT). This work was also PR:FUNDING_SOURCE developed within the scope of the project CICECO-Aveiro Institute of Materials, PR:FUNDING_SOURCE UIDB/50011/2020 (doi: 10.54499/UIDB/50011/2020), UIDB/50011/2020, PR:FUNDING_SOURCE UIDP/50011/2020 (doi: 10.54499/UIDP/50011/2020) & LA/P/0006/2020, financed by PR:FUNDING_SOURCE national funds through the FCT/MCTES (PIDDAC). The NMR spectrometer is part of PR:FUNDING_SOURCE the National NMR Network (PTNMR) and are partially supported by Infrastructure PR:FUNDING_SOURCE Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and PR:FUNDING_SOURCE FCT through PIDDAC). T.N. acknowledges FCT Individual Call to Scientific PR:FUNDING_SOURCE Employment Stimulus 2022 (2022.02925.CEECIND/CP1728/CT0004, doi: 10.54499/ PR:FUNDING_SOURCE 2022.02925.CEECIND/CP1728/CT0004). #STUDY ST:STUDY_TITLE 1H NMR metabolomics applied to assess the direct and transgenerational effects ST:STUDY_TITLE of simvastatin on the metabolism of the amphipod Gammarus locusta ST:STUDY_TYPE 1H NMR metabolomics to study the metabolic effects of direct exposure to ST:STUDY_TYPE simvastatin and transgenerational effects on the polar metabolome of amphipod ST:STUDY_TYPE Gammarus locusta ST:STUDY_SUMMARY In this study, a comprehensive untargeted 1H NMR metabolomics strategy was ST:STUDY_SUMMARY applied to measure the metabolic impact of direct and transgenerational exposure ST:STUDY_SUMMARY (F0 and F3 generations, respectively) of amphipods G. locusta to simvastatin ST:STUDY_SUMMARY (SIM), a pharmaceutical widely prescribed for the treatment of ST:STUDY_SUMMARY hypercholesterolemia. To assess the direct and transgenerational effects of ST:STUDY_SUMMARY exposure to SIM on the metabolism of G. locusta, the following conditions were ST:STUDY_SUMMARY studied: i) direct exposure of parental generation (F0) of males and females, ST:STUDY_SUMMARY GM/GF-F0 (Exp); and ii) transgenerational effects, considering the first ST:STUDY_SUMMARY unexposed generation (F3) of males and females, to assess transgenerational ST:STUDY_SUMMARY effects, GM/GF-F3 (Tr). The obtained data added important knowledge, unveiling ST:STUDY_SUMMARY individual metabolic effects of direct exposure to simvastatin and its ST:STUDY_SUMMARY transgenerational effects, potentially contributing towards improving hazard and ST:STUDY_SUMMARY risk assessment of biologically active compounds. ST:INSTITUTE University of Aveiro ST:DEPARTMENT CICECO – Aveiro Institute of Materials, Department of Chemistry ST:LABORATORY Metabolomics Group ST:LAST_NAME Rodrigues ST:FIRST_NAME João A. ST:ADDRESS University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, ST:ADDRESS Portugal ST:EMAIL joao.rodrigues@ua.pt ST:PHONE 00351234370707 ST:NUM_GROUPS 8 ST:TOTAL_SUBJECTS 39 ST:NUM_MALES 20 ST:NUM_FEMALES 19 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Gammarus locusta SU:TAXONOMY_ID 65426 SU:WEIGHT_OR_WEIGHT_RANGE length: 14-16 and 10-12.5 mm for males and females, respectively (whole body) SU:GENDER Male and female SU:SPECIES_GROUP Gammarus locusta #FACTORS #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 GF-F0_Ctr GF-F0_Ctr_01 Sample source:whole_body | Factor:GF-F0_Ctr RAW_FILE_NAME=gammarus_001.raw SUBJECT_SAMPLE_FACTORS GF-F0_Ctr GF-F0_Ctr_02 Sample source:whole_body | Factor:GF-F0_Ctr RAW_FILE_NAME=gammarus_002.raw SUBJECT_SAMPLE_FACTORS GF-F0_Ctr GF-F0_Ctr_03 Sample source:whole_body | Factor:GF-F0_Ctr RAW_FILE_NAME=gammarus_003.raw SUBJECT_SAMPLE_FACTORS GF-F0_Ctr GF-F0_Ctr_04 Sample source:whole_body | Factor:GF-F0_Ctr RAW_FILE_NAME=gammarus_004.raw SUBJECT_SAMPLE_FACTORS GF-F0_Ctr GF-F0_Ctr_05 Sample source:whole_body | Factor:GF-F0_Ctr RAW_FILE_NAME=gammarus_005.raw SUBJECT_SAMPLE_FACTORS GF-F0_Exp GF-F0_Exp_06 Sample source:whole_body | Factor:GF-F0_Exp RAW_FILE_NAME=gammarus_006.raw SUBJECT_SAMPLE_FACTORS GF-F0_Exp GF-F0_Exp_07 Sample source:whole_body | Factor:GF-F0_Exp RAW_FILE_NAME=gammarus_007.raw SUBJECT_SAMPLE_FACTORS GF-F0_Exp GF-F0_Exp_08 Sample source:whole_body | Factor:GF-F0_Exp RAW_FILE_NAME=gammarus_008.raw SUBJECT_SAMPLE_FACTORS GF-F0_Exp GF-F0_Exp_09 Sample source:whole_body | Factor:GF-F0_Exp RAW_FILE_NAME=gammarus_009.raw SUBJECT_SAMPLE_FACTORS GF-F0_Exp GF-F0_Exp_10 Sample source:whole_body | Factor:GF-F0_Exp RAW_FILE_NAME=gammarus_010.raw SUBJECT_SAMPLE_FACTORS GF-F3_Ctr GF-F3_Ctr_01 Sample source:whole_body | Factor:GF-F3_Ctr RAW_FILE_NAME=gammarus_011.raw SUBJECT_SAMPLE_FACTORS GF-F3_Ctr GF-F3_Ctr_02 Sample source:whole_body | Factor:GF-F3_Ctr RAW_FILE_NAME=gammarus_012.raw SUBJECT_SAMPLE_FACTORS GF-F3_Ctr GF-F3_Ctr_03 Sample source:whole_body | Factor:GF-F3_Ctr RAW_FILE_NAME=gammarus_013.raw SUBJECT_SAMPLE_FACTORS GF-F3_Ctr GF-F3_Ctr_04 Sample source:whole_body | Factor:GF-F3_Ctr RAW_FILE_NAME=gammarus_014.raw SUBJECT_SAMPLE_FACTORS GF-F3_Tr GF-F3_Tr_06 Sample source:whole_body | Factor:GF-F3_Tr RAW_FILE_NAME=gammarus_015.raw SUBJECT_SAMPLE_FACTORS GF-F3_Tr GF-F3_Tr_07 Sample source:whole_body | Factor:GF-F3_Tr RAW_FILE_NAME=gammarus_016.raw SUBJECT_SAMPLE_FACTORS GF-F3_Tr GF-F3_Tr_08 Sample source:whole_body | Factor:GF-F3_Tr RAW_FILE_NAME=gammarus_017.raw SUBJECT_SAMPLE_FACTORS GF-F3_Tr GF-F3_Tr_09 Sample source:whole_body | Factor:GF-F3_Tr RAW_FILE_NAME=gammarus_018.raw SUBJECT_SAMPLE_FACTORS GF-F3_Tr GF-F3_Tr_10 Sample source:whole_body | Factor:GF-F3_Tr RAW_FILE_NAME=gammarus_019.raw SUBJECT_SAMPLE_FACTORS GM-F0_Ctr GM-F0_Ctr_01 Sample source:whole_body | Factor:GM-F0_Ctr RAW_FILE_NAME=gammarus_020.raw SUBJECT_SAMPLE_FACTORS GM-F0_Ctr GM-F0_Ctr_02 Sample source:whole_body | Factor:GM-F0_Ctr RAW_FILE_NAME=gammarus_021.raw SUBJECT_SAMPLE_FACTORS GM-F0_Ctr GM-F0_Ctr_03 Sample source:whole_body | Factor:GM-F0_Ctr RAW_FILE_NAME=gammarus_022.raw SUBJECT_SAMPLE_FACTORS GM-F0_Ctr GM-F0_Ctr_04 Sample source:whole_body | Factor:GM-F0_Ctr RAW_FILE_NAME=gammarus_023.raw SUBJECT_SAMPLE_FACTORS GM-F0_Ctr GM-F0_Ctr_05 Sample source:whole_body | Factor:GM-F0_Ctr RAW_FILE_NAME=gammarus_024.raw SUBJECT_SAMPLE_FACTORS GM-F0_Exp GM-F0_Exp_06 Sample source:whole_body | Factor:GM-F0_Exp RAW_FILE_NAME=gammarus_025.raw SUBJECT_SAMPLE_FACTORS GM-F0_Exp GM-F0_Exp_07 Sample source:whole_body | Factor:GM-F0_Exp RAW_FILE_NAME=gammarus_026.raw SUBJECT_SAMPLE_FACTORS GM-F0_Exp GM-F0_Exp_08 Sample source:whole_body | Factor:GM-F0_Exp RAW_FILE_NAME=gammarus_027.raw SUBJECT_SAMPLE_FACTORS GM-F0_Exp GM-F0_Exp_09 Sample source:whole_body | Factor:GM-F0_Exp RAW_FILE_NAME=gammarus_028.raw SUBJECT_SAMPLE_FACTORS GM-F0_Exp GM-F0_Exp_10 Sample source:whole_body | Factor:GM-F0_Exp RAW_FILE_NAME=gammarus_029.raw SUBJECT_SAMPLE_FACTORS GM-F3_Ctr GM-F3_Ctr_01 Sample source:whole_body | Factor:GM-F3_Ctr RAW_FILE_NAME=gammarus_030.raw SUBJECT_SAMPLE_FACTORS GM-F3_Ctr GM-F3_Ctr_02 Sample source:whole_body | Factor:GM-F3_Ctr RAW_FILE_NAME=gammarus_031.raw SUBJECT_SAMPLE_FACTORS GM-F3_Ctr GM-F3_Ctr_03 Sample source:whole_body | Factor:GM-F3_Ctr RAW_FILE_NAME=gammarus_032.raw SUBJECT_SAMPLE_FACTORS GM-F3_Ctr GM-F3_Ctr_04 Sample source:whole_body | Factor:GM-F3_Ctr RAW_FILE_NAME=gammarus_033.raw SUBJECT_SAMPLE_FACTORS GM-F3_Ctr GM-F3_Ctr_05 Sample source:whole_body | Factor:GM-F3_Ctr RAW_FILE_NAME=gammarus_034.raw SUBJECT_SAMPLE_FACTORS GM-F3_Tr GM-F3_Tr_06 Sample source:whole_body | Factor:GM-F3_Tr RAW_FILE_NAME=gammarus_035.raw SUBJECT_SAMPLE_FACTORS GM-F3_Tr GM-F3_Tr_07 Sample source:whole_body | Factor:GM-F3_Tr RAW_FILE_NAME=gammarus_036.raw SUBJECT_SAMPLE_FACTORS GM-F3_Tr GM-F3_Tr_08 Sample source:whole_body | Factor:GM-F3_Tr RAW_FILE_NAME=gammarus_037.raw SUBJECT_SAMPLE_FACTORS GM-F3_Tr GM-F3_Tr_09 Sample source:whole_body | Factor:GM-F3_Tr RAW_FILE_NAME=gammarus_038.raw SUBJECT_SAMPLE_FACTORS GM-F3_Tr GM-F3_Tr_10 Sample source:whole_body | Factor:GM-F3_Tr RAW_FILE_NAME=gammarus_039.raw #COLLECTION CO:COLLECTION_SUMMARY To assess the direct and transgenerational effects of exposure to SIM on the CO:COLLECTION_SUMMARY metabolism of G. locusta, the following conditions were studied: i) direct CO:COLLECTION_SUMMARY exposure of parental generation (F0) of males and females, GM/GF-F0 (Exp); and CO:COLLECTION_SUMMARY ii) transgenerational effects, considering the first unexposed generation (F3) CO:COLLECTION_SUMMARY of males and females, to assess transgenerational effects, GM/GF-F3 (Tr). The CO:COLLECTION_SUMMARY experimental protocol followed an earlier report (Neuparth et al., 2020). Direct CO:COLLECTION_SUMMARY exposure (F0 generation) experiments involved the selection of 50 one-week-old CO:COLLECTION_SUMMARY G. locusta offspring randomly placed in 7 L aquaria. Each aquarium was filled CO:COLLECTION_SUMMARY with natural filtered seawater (5 L) defining the following two groups: i) CO:COLLECTION_SUMMARY exposed to a SIM (320 ng/L) solution in 0.0005% acetone, GM/GF-F0 (Exp) group; CO:COLLECTION_SUMMARY and ii) controls, in filtered natural seawater with 0.0005% acetone, GM/GF-F0 CO:COLLECTION_SUMMARY (Ct) group. The chosen SIM concentration (320 ng/L) was based on concentrations CO:COLLECTION_SUMMARY found/estimated in Portuguese wastewaters. In each aquarium, a sediment layer of CO:COLLECTION_SUMMARY ca. 1 cm and several small stones were placed to simulate the species’ natural CO:COLLECTION_SUMMARY habitat. G. locusta individuals were kept in filtered natural saltwater CO:COLLECTION_SUMMARY (33–35‰ salinity), at 20 °C, under a 16:8h (light: dark) photoperiod, and CO:COLLECTION_SUMMARY were fed ad libitum with the macroalgae Ulva sp.. The water was renewed every 3 CO:COLLECTION_SUMMARY days, with the SIM concentration being re-established by spiking each aquarium CO:COLLECTION_SUMMARY with a standard solution. The actual SIM concentration was monitored during the CO:COLLECTION_SUMMARY assay. G. locusta individuals were kept under the conditions above for 55 days CO:COLLECTION_SUMMARY (until adulthood), after which 5 males (GM) and 5 females (GF) were randomly CO:COLLECTION_SUMMARY collected (whole body, 14-16 and 10-12.5 mm in length, for males and females CO:COLLECTION_SUMMARY respectively) from each condition and stored at - 80°C until tissue extraction. CO:COLLECTION_SUMMARY To evaluate the transgenerational effects of SIM exposure, the offspring of the CO:COLLECTION_SUMMARY F0 generation organisms (exposed to SIM) were placed in SIM-free water (natural CO:COLLECTION_SUMMARY filtered seawater) during three consecutive generations (F1, F2 and F3), giving CO:COLLECTION_SUMMARY rise to the group designated as GM/GF-F3 (Tr) (Figure 1). The corresponding CO:COLLECTION_SUMMARY control group comprised the F3 generation of the individuals previously CO:COLLECTION_SUMMARY unexposed to SIM, designated as GM/GF-F3 (Ct). For each condition, 4 replicate CO:COLLECTION_SUMMARY aquaria were used. Each generation was started with 50 offspring of the previous CO:COLLECTION_SUMMARY one, and kept for 55-65 days, to achieve adulthood. At the F3 generation, 5 CO:COLLECTION_SUMMARY males (GM) and 5 females (GF) (measuring 14-16 and 10-12.5 mm in length, CO:COLLECTION_SUMMARY respectively) were randomly collected for transgenerational, F3 (Tr), and CO:COLLECTION_SUMMARY control groups, F3 (Ct), and stored at -80°C until tissue extraction. CO:COLLECTION_PROTOCOL_FILENAME 1. Gammarus experimental procedure CO:SAMPLE_TYPE Whole animals CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY To assess the direct and transgenerational effects of exposure to SIM on the TR:TREATMENT_SUMMARY metabolism of G. locusta, the following conditions were studied: i) direct TR:TREATMENT_SUMMARY exposure of parental generation (F0) of males and females, GM/GF-F0 (Exp); and TR:TREATMENT_SUMMARY ii) transgenerational effects, considering the first unexposed generation (F3) TR:TREATMENT_SUMMARY of males and females, to assess transgenerational effects, GM/GF-F3 (Tr). Direct TR:TREATMENT_SUMMARY exposure (F0 generation) experiments involved the selection of 50 one-week-old TR:TREATMENT_SUMMARY G. locusta offspring randomly placed in 7 L aquaria. Each aquarium was filled TR:TREATMENT_SUMMARY with natural filtered seawater (5 L) defining the following two groups: i) TR:TREATMENT_SUMMARY exposed to a SIM (320 ng/L) solution in 0.0005% acetone, GM/GF-F0 (Exp) group; TR:TREATMENT_SUMMARY and ii) controls, in filtered natural seawater with 0.0005% acetone, GM/GF-F0 TR:TREATMENT_SUMMARY (Ct) group. The chosen SIM concentration (320 ng/L) was based on concentrations TR:TREATMENT_SUMMARY found/estimated in Portuguese wastewaters. In each aquarium, a sediment layer of TR:TREATMENT_SUMMARY ca. 1 cm and several small stones were placed to simulate the species’ natural TR:TREATMENT_SUMMARY habitat. G. locusta individuals were kept in filtered natural saltwater TR:TREATMENT_SUMMARY (33–35‰ salinity), at 20 °C, under a 16:8h (light: dark) photoperiod, and TR:TREATMENT_SUMMARY were fed ad libitum with the macroalgae Ulva sp.. The water was renewed every 3 TR:TREATMENT_SUMMARY days, with the SIM concentration being re-established by spiking each aquarium TR:TREATMENT_SUMMARY with a standard solution. The actual SIM concentration was monitored during the TR:TREATMENT_SUMMARY assay. G. locusta individuals were kept under the conditions above for 55 days TR:TREATMENT_SUMMARY (until adulthood), after which 5 males (GM) and 5 females (GF) were randomly TR:TREATMENT_SUMMARY collected (whole body, 14-16 and 10-12.5 mm in length, for males and females TR:TREATMENT_SUMMARY respectively) from each condition and stored at - 80°C until tissue extraction. TR:TREATMENT_SUMMARY To evaluate the transgenerational effects of SIM exposure, the offspring of the TR:TREATMENT_SUMMARY F0 generation organisms (exposed to SIM) were placed in SIM-free water (natural TR:TREATMENT_SUMMARY filtered seawater) during three consecutive generations (F1, F2 and F3), giving TR:TREATMENT_SUMMARY rise to the group designated as GM/GF-F3 (Tr) (Figure 1). The corresponding TR:TREATMENT_SUMMARY control group comprised the F3 generation of the individuals previously TR:TREATMENT_SUMMARY unexposed to SIM, designated as GM/GF-F3 (Ct). For each condition, 4 replicate TR:TREATMENT_SUMMARY aquaria were used. Each generation was started with 50 offspring of the previous TR:TREATMENT_SUMMARY one, and kept for 55-65 days, to achieve adulthood. At the F3 generation, 5 TR:TREATMENT_SUMMARY males (GM) and 5 females (GF) (measuring 14-16 and 10-12.5 mm in length, TR:TREATMENT_SUMMARY respectively) were randomly collected for transgenerational, F3 (Tr), and TR:TREATMENT_SUMMARY control groups, F3 (Ct), and stored at -80°C until tissue extraction. TR:TREATMENT_PROTOCOL_FILENAME 1. Gammarus experimental procedure TR:TREATMENT_COMPOUND Direct (F0 generation) and transgenerational (F3 generation) exposure to TR:TREATMENT_COMPOUND simvastatin TR:TREATMENT_DOSE G. locusta animals were: i) directly exposed to 320 ng/L of simvastatin for 55 TR:TREATMENT_DOSE days (until adulthood); and ii) transgenerational exposed to simvastatin by the TR:TREATMENT_DOSE offspring of the F0 generation organisms being exposed to 320 ng/L of TR:TREATMENT_DOSE simvastatin for 55 days (F0 generations) and the following generations were TR:TREATMENT_DOSE placed in a simvastatin-free water (natural filtered seawater) during three TR:TREATMENT_DOSE consecutive generations (F1, F2 and F3). TR:TREATMENT_DOSEDURATION 55 days for direct exposure; 220 days for transgenerational exposure #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolite extraction was carried out using a water/methanol/chloroform method, SP:SAMPLEPREP_SUMMARY 0.75:1:1 (v/v/v), as described elsewhere (Hines, Oladiran, Bignell et al., SP:SAMPLEPREP_SUMMARY 2007). Briefly, each frozen G. locusta individual was placed in 2 mL Precellys SP:SAMPLEPREP_SUMMARY tubes with 150 mg glass beads (ø = 0.5 mm). A volume of 750 μL of cold SP:SAMPLEPREP_SUMMARY methanol: ultrapure water (4:1) solution was added before homogenization with a SP:SAMPLEPREP_SUMMARY Precellys Evolution Touch homogenizer (Bertin Technologies, SP:SAMPLEPREP_SUMMARY Montigny-le-Bretonneux, France) (two cycles of 10 s homogenization at 6500 rpm, SP:SAMPLEPREP_SUMMARY with 15 s of rest between cycles). Subsequently, 300 µL cold chloroform was SP:SAMPLEPREP_SUMMARY added and samples were vortexed, followed by addition of 300 µL cold chloroform SP:SAMPLEPREP_SUMMARY and 300 µL cold ultrapure water. After 10 min at -20 °C, samples were SP:SAMPLEPREP_SUMMARY centrifuged (2,500 g, 4 °C, 10 min) and the upper (polar) layer was transferred SP:SAMPLEPREP_SUMMARY into vials, dried in a centrifugal vacuum concentrator (UNIVAP 100H) and stored SP:SAMPLEPREP_SUMMARY at -80 °C until NMR analysis. SP:SAMPLEPREP_PROTOCOL_FILENAME 1. Gammarus experimental procedure SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Water/methanol/chloroform method, as described in (Hines, Oladiran, Bignell et SP:EXTRACTION_METHOD al., 2007) SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION The dried polar extracts of G. locusta samples were re-suspended in 600 μL of SP:SAMPLE_RESUSPENSION 100 mM sodium phosphate buffer (pH 7.4), in D2O (99.9% deuterium) containing 0.5 SP:SAMPLE_RESUSPENSION mM sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP-d4, in D2O, SP:SAMPLE_RESUSPENSION for chemical shift referencing). After vortex homogenization and centrifugation SP:SAMPLE_RESUSPENSION (16,000 g, room temperature, 10 min), 550 μL of the supernatant were SP:SAMPLE_RESUSPENSION transferred to 5 mm NMR tubes. SP:SAMPLE_SPIKING 0.5 mM sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP-d4), as a SP:SAMPLE_SPIKING chemical shift reference. #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME Metabolomics Group AN:OPERATOR_NAME Joao A. Rodrigues AN:DETECTOR_TYPE NMR Bruker Avance III 500 MHz AN:SOFTWARE_VERSION TopSpin3.2 and Amix3.9.14 #NMR NM:INSTRUMENT_NAME Bruker Avance III NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 500 MHz NM:NMR_PROBE TXI NM:NMR_SOLVENT 100 mM sodium phosphate buffer (pH 7.4), in D2O (99.9% deuterium) containing 0.5 NM:NMR_SOLVENT mM sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP-d4, in D2O, NM:NMR_SOLVENT for chemical shift referencing). NM:NMR_TUBE_SIZE 5 mm NMR tubes NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE noesypr1d NM:WATER_SUPPRESSION presat NM:PULSE_WIDTH 90-degree NM:RECEIVER_GAIN 203 NM:OFFSET_FREQUENCY 2352 Hz NM:CHEMICAL_SHIFT_REF_CPD 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP-d4) NM:TEMPERATURE 298 K NM:NUMBER_OF_SCANS 256 scans NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 2.33 s NM:RELAXATION_DELAY 5 s NM:SPECTRAL_WIDTH 7,002.8 Hz NM:NUM_DATA_POINTS_ACQUIRED 32 k points NM:LINE_BROADENING 0.3 Hz NM:ZERO_FILLING 64 k points NM:APODIZATION Exponential NM:BASELINE_CORRECTION_METHOD Manual NM:CHEMICAL_SHIFT_REF_STD 0 ppm for TSP-d4 NM:NMR_RESULTS_FILE ST003473_AN005708_Results.txt UNITS:Intensity #END