Summary of Study ST000049

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR000024. The data can be accessed directly via it's Project DOI: 10.21228/M85P4V This work is supported by NIH grant, U2C- DK119886.


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Study IDST000049
Study TitleMetabolomics Analysis of Thermally Challenged Mayfly Larvae (NMR analysis)
Study TypeMetabolomic analysis of mayflies
Study SummaryThe purpose of this study was to examine the metabolic profiles of mayfly (Centroptilum triangulifer) larvae subjected to thermal challenge. This species is unusual in terms of its ease of culture, and its suitability as a laboratory test organism. Our purpose here was to examine how an environmentally realistic thermal challenge affects the physiology of this organism. In this study, we obtained several types of insect species and we were able to show that NMR Metabolomics could be used to distinguish among the different types of larvae.
University of North Carolina
DepartmentDiscovery Science Technology
LaboratorySumner Lab
Last NameSumner
First NameSusan
AddressEastern Regional Comprehensive Metabolomics Resource Core, UNC Nutrition Research Institute, 500 Laureate Way, Kannapolis, NC, 28081
Submit Date2014-02-28
Num Groups7
Raw Data AvailableYes
Raw Data File Type(s)fid
Uploaded File Size7.6 M
Analysis Type DetailNMR
Release Date2015-05-01
Release Version1
Susan Sumner Susan Sumner application/zip

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Project ID:PR000024
Project DOI:doi: 10.21228/M85P4V
Project Title:Metabolomics Analysis of Thermally Challenged Mayfly Larvae
Project Type:MS/NMR
Project Summary:The purpose of this study was to examine the metabolic profiles of mayfly (Centroptilum triangulifer) larvae subjected to a thermal challenge. This species is unusual in terms of its ease of culture, and its suitability as a laboratory test organism. Studies complementary to this metabolic study have focused on thermal effects on metabolic rates (respiration) and gene expression patterns in this species and do not support Prtners oxygen limitation paradigm. Our purpose here was to examine how an environmentally realistic thermal challenge affects the physiology of this organism.
Institute:North Carolina State University
Department:Environmental and Molecular Toxicology
Laboratory:Buchwalter Laboratory
Last Name:Buchwalter
First Name:David
Address:North Carolina State University Raleigh, NC 27695


Subject ID:SU000067
Subject Type:Insect
Subject Species:Centroptilum triangulifer;Atherix;Megaloptera
Taxonomy ID:2078957;124299;50553
Species Group:Insect


Subject type: Insect; Subject species: Centroptilum triangulifer;Atherix;Megaloptera (Factor headings shown in green)

mb_sample_id local_sample_id species Treatment wing pads
SA002143AD_01Athorix Diptoa Athorix Diptoa control no wing pads
SA002144Pooled_QC_01Mayfly, Megalopterans, Athorix Diptoa Pooled QC sample no wing pads
SA002145Pooled_QC_02Mayfly, Megalopterans, Athorix Diptoa Pooled QC sample no wing pads
SA002146Pooled_QC_03Mayfly, Megalopterans, Athorix Diptoa Pooled QC sample no wing pads
SA002150MF_01Mayfly Mayfly control from different study no wing pads
SA002152Control_02Mayfly Mayfly control no wing pads
SA002153Control_03Mayfly Mayfly control no wing pads
SA002154Control_01Mayfly Mayfly control no wing pads
SA002151Control_XMayfly Mayfly controlX wing pads
SA002147Heat_Treated_03Mayfly Mayfly Heat Treated no wing pads
SA002148Heat_Treated_02Mayfly Mayfly Heat Treated no wing pads
SA002149Heat_Treated_01Mayfly Mayfly Heat Treated no wing pads
SA002155MG_01Megalopterans Megalopterans control no wing pads
Showing results 1 to 13 of 13


Collection ID:CO000050
Collection Summary:whole insects were collected
Collection Protocol Comments:Original culture material was obtained from the Stroud Water Research Center (Avondale, PA). Larvae were reared at 22°C and fed a periphytic diet. When larvae reached a suitable size, they were separated from their food source for 12 hours to evacuate gut contents. Larvae were then divided into two treatment groups – a control group maintained at the culturing temperature of 22°C, and a thermal challenge group. The thermal challenge group was subjected to a temperature increase at a rate of 1°C per hour. This rate of temperature change is commonly observed in temperate streams. When the treatment temperature reached 30°C, larvae from both control and treatment groups were flash frozen in liquid nitrogen in groups of 12-13 larvae. Each grouping of 12-13 larvae comprised a composite replicate for the metabolic profiling analysis. A total of 3 replicates each for the control and thermal treatments were used in this study. Some larvae had developed dark wing pads during the experiment. This is a signal that the larvae were very close to emerging from the aquatic larval phase to a winged sub-adult phase. Enough of these larvae were present in the control treatment to be removed from the control cohort and considered as an independent sample.
Sample Type:whole insect
Volumeoramount Collected:50 mg
Storage Conditions:-80C


Treatment ID:TR000068
Treatment Summary:Metabolomics profile of mayfly larvae exposed to heat
Treatment Protocol Comments:Heat Treated | Control | ControlX
Animal Acclimation Duration:exposed to heat (22C to 30C ramp); fasted for 8 hours | control (fasted for 8 hours) | control (fasted for 6 hours)

Sample Preparation:

Sampleprep ID:SP000063
Sampleprep Summary:Control and heat-treated mayfly samples were provided in triplicate and were processed individually. One sample was provided for AD, ControlX, MF control (from a previous study) and MG larvae. Three replicates of the larvae listed above having only a single sample were created. Aliquots of 50-300mg of insect larvae were mixed with degassed 1:1 Acetonitrile:Water solution in a 2 mL snap cap tube at a concentration of 50 mg/mL. Megalopterans were substantially larger than the other larvae and were prepared at 200 mg/mL. Samples were homogenized and then centrifuged at 4?C for 5 minutes at 14000rcf. The supernatant was removed and placed into a new tube. Samples were centrifuged again at 4?C for 5 minutes at 14000rcf and a volume of the homogenate corresponding to 40 mg insect larvae were taken. After homogenization, the three larval replicates from AD, ControlX, MG and MF control (from a previous study) were pooled and a single sample created for each larval type. There was insufficient Mayfly (heat and control) sample material to create an internal pool, so an external pool was made by combining equal concentration amounts of AD, MG and MF control (from a previous study) pool quality check (QC) samples. Samples were completely dried by vacuum centrifuge and were reconstituted by adding 630 µL of D2O (Aldrich) and 70 µL of Chenomx ISTD solution (Chenomx, Edmonton, Alberta, Canada). The samples were vortexed and centrifuged at 14000rcf for 2 minutes. 650 µL of sample was transferred into 5mm NMR tubes and analyzed by NMR.
Sampleprep Protocol Filename:RTI_Metabolomic_Analysis_of_Thermally_Challenged_Larvae_Procedure.docx
Processing Method:Homogenization and Solvent Removal with SpeedVac
Processing Storage Conditions:4°C
Extraction Method:50% Aqueous Acetonitrile
Extract Concentration Dilution:dried on SpeedVac
Extract Storage:-80C
Sample Resuspension:resuspended in D2O + Chenomx ISTD solution


Analysis ID:AN000085
Laboratory Name:DHMRI
Analysis Type:NMR
Acquisition Date:41624
Software Version:TopSpin 3.0
Operator Name:Kevin Knagge, Wimal Pathmasiri
Randomization Order:Yes
Detector Type:NMR
Data Format:Bruker
Chromatography ID:CH000054
Num Factors:7


NMR ID:NM000022
Analysis ID:AN000085
Instrument Name:Bruker Avance III
Instrument Type:FT-NMR
NMR Experiment Type:1D 1H
Field Frequency Lock:D20
Standard Concentration:0.5 mM
Spectrometer Frequency:950 MHz
NMR Probe:5mm Cryogenically Cooled ATMA
NMR Solvent:D20
NMR Tube Size:5mm
Shimming Method:Topshim (Gradient)
Pulse Sequence:noesypr1d
Water Suppression:yes
Pulse Width:8.68
Power Level:12.589W
Receiver Gain:4
Offset Frequency:4468.30 Hz
Chemical Shift Ref Cpd:DSS
Temperature:298 K
Number Of Scans:256
Dummy Scans:4
Acquisition Time:1.73 sec
Relaxation Delay:2s
Spectral Width:18939.395 Hz
Num Data Points Acquired:65536
Real Data Points:65536
Line Broadening:0.5
Zero Filling:Yes
Baseline Correction Method:Polynomial
Chemical Shift Ref Std:DSS