#METABOLOMICS WORKBENCH jroth002_20190531_133902 DATATRACK_ID:1730 STUDY_ID:ST001187 ANALYSIS_ID:AN001979 PROJECT_ID:PR000799 VERSION 1 CREATED_ON June 2, 2019, 12:30 pm #PROJECT PR:PROJECT_TITLE Untargeted metabolomics of honey bees exposed to selenate or cadmium PR:PROJECT_SUMMARY Effects of selenate and cadmium exposure on honey bees. PR:INSTITUTE UC Riverside PR:LAST_NAME Rothman PR:FIRST_NAME Jason PR:ADDRESS 900 University Ave., Riverside, CA, 91766, USA PR:EMAIL jroth002@ucr.edu PR:PHONE 9518275817 #STUDY ST:STUDY_TITLE Effects of selenate and cadmium exposure on the honey bee metabolome ST:STUDY_SUMMARY We moved one frame of brood each from five healthy honey bee colonies with ST:STUDY_SUMMARY marked Italian queens and housed them in a hive body at 35°C and 50% humidity ST:STUDY_SUMMARY under constant darkness. We then allowed the bees to emerge, mixed the newly ST:STUDY_SUMMARY emerged workers (NEWs) to randomize their colony of origin and placed NEWs into ST:STUDY_SUMMARY 13 cm x 10.5 cm x 6.5 cm wire cages equipped with feeders containing 35mL of ST:STUDY_SUMMARY deionized water and 35mL 50% sucrose. We also provided a pollen patty to each ST:STUDY_SUMMARY cage of bees consisting of 269g corn syrup, 113g sucrose and 113g of Bee Pro ST:STUDY_SUMMARY (Mann Lake, Hackensack, MN). To inoculate the newly emerged workers with their ST:STUDY_SUMMARY “core” microbiome, we collected 50 mL of foragers from the source hives of ST:STUDY_SUMMARY the NEWs, immobilized the bees at 4°C, aseptically dissected out the abdomens ST:STUDY_SUMMARY and macerated the whole abdomens in 50% sucrose. We added 1 mL of the resulting ST:STUDY_SUMMARY slurry to 34 mL of 50% sucrose solution and fed it to the NEWs. We allowed the ST:STUDY_SUMMARY bees to feed ad libitium on the mixture for two days before replacing the ST:STUDY_SUMMARY feeders with 50% sucrose. We allowed the bees to feed for three more days to ST:STUDY_SUMMARY fully establish a microbiome. Once the bees had an established microbiome, we ST:STUDY_SUMMARY prepared treatment feeding solutions of 50% sucrose (as a no metal/metalloid ST:STUDY_SUMMARY control), 50% sucrose spiked with 0.6 mg/L sodium selenate or 50% sucrose with ST:STUDY_SUMMARY 0.24 mg/L cadmium chloride (Alfa Aesar, Ward Hill, MA) and pollen patties spiked ST:STUDY_SUMMARY with either 6.0 mg/L selenium or 0.46 mg/L cadmium as in Hladun et al 2015. We ST:STUDY_SUMMARY again allowed the bees to feed ad libitium. We sampled three bees from 13 cages ST:STUDY_SUMMARY after four days of continuous exposure to the above-mentioned treatments and ST:STUDY_SUMMARY immediately placed the samples on dry ice, followed by long-term storage at -80 ST:STUDY_SUMMARY °C. ST:INSTITUTE UC Riverside ST:LAST_NAME Rothman ST:FIRST_NAME Jason ST:ADDRESS 900 University Ave. ST:EMAIL jroth002@ucr.edu ST:PHONE 9518275817 #SUBJECT SU:SUBJECT_TYPE Insect SU:SUBJECT_SPECIES Apis mellifera SU:TAXONOMY_ID 7460 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - QC-1 Treatment:Pool SUBJECT_SAMPLE_FACTORS - HBCT-4 Treatment:Control SUBJECT_SAMPLE_FACTORS - HBSE-2 Treatment:Selenate SUBJECT_SAMPLE_FACTORS - HBSE-4 Treatment:Selenate SUBJECT_SAMPLE_FACTORS - QC-2 Treatment:Pool SUBJECT_SAMPLE_FACTORS - HBSE-5 Treatment:Selenate SUBJECT_SAMPLE_FACTORS - HBCT-3 Treatment:Control SUBJECT_SAMPLE_FACTORS - HBCD-3 Treatment:Cadmium SUBJECT_SAMPLE_FACTORS - QC-3 Treatment:Pool SUBJECT_SAMPLE_FACTORS - HBSE-1 Treatment:Selenate SUBJECT_SAMPLE_FACTORS - HBCT-1 Treatment:Control SUBJECT_SAMPLE_FACTORS - HBCD-4 Treatment:Cadmium SUBJECT_SAMPLE_FACTORS - HBCT-2 Treatment:Control SUBJECT_SAMPLE_FACTORS - QC-4 Treatment:Pool SUBJECT_SAMPLE_FACTORS - HBCD-2 Treatment:Cadmium SUBJECT_SAMPLE_FACTORS - HBSE-3 Treatment:Selenate SUBJECT_SAMPLE_FACTORS - HBCD-1 Treatment:Cadmium SUBJECT_SAMPLE_FACTORS - QC-5 Treatment:Pool #COLLECTION CO:COLLECTION_SUMMARY We sampled three bees from 13 cages after four days of continuous exposure to CO:COLLECTION_SUMMARY the treatments and immediately placed the samples on dry ice, followed by CO:COLLECTION_SUMMARY long-term storage at -80 °C. CO:SAMPLE_TYPE Insect tissue CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY Once the bees had an established microbiome, we prepared treatment feeding TR:TREATMENT_SUMMARY solutions of 50% sucrose (as a no metal/metalloid control), 50% sucrose spiked TR:TREATMENT_SUMMARY with 0.6 mg/L sodium selenate or 50% sucrose with 0.24 mg/L cadmium chloride TR:TREATMENT_SUMMARY (Alfa Aesar, Ward Hill, MA) and pollen patties spiked with either 6.0 mg/L TR:TREATMENT_SUMMARY selenium or 0.46 mg/L cadmium and allowed the bees to feed ad libitium. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were freeze-dried, homogenized, and extracted with 30:30:20:20 SP:SAMPLEPREP_SUMMARY acetonitrile:methanol:water:isopropanol. Samples were then sonicated, vortexed, SP:SAMPLEPREP_SUMMARY and centrifuged before analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Normal phase CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Waters Acquity CSH C18 (100 x 2.1mm, 1.7um) CH:FLOW_GRADIENT 0 min, 1% B; 1 min, 1% B; 8 min, 40% B; 24 min, 100% B; 26.5 min, 100% B; 27 CH:FLOW_GRADIENT min, 1% B CH:FLOW_RATE 250 ul/min CH:COLUMN_TEMPERATURE 40 CH:SOLVENT_A 0.1% formic acid CH:SOLVENT_B Acetonitrile with 0.1% formic acid #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME UC Riverside Metabolomics Core Facility #MS MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS We processed the metabolite data (peak picking, alignment, deconvolution, MS:MS_COMMENTS integration, normalization, and spectral matching) with Progenesis Qi software MS:MS_COMMENTS (Nonlinear Dynamics, Durham, NC). MS:MS_RESULTS_FILE ST001187_AN001979_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END