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MB Sample ID: SA002148
| Local Sample ID: | Heat_Treated_02 |
| Subject ID: | SU000067 |
| Subject Type: | Insect |
| Subject Species: | Centroptilum triangulifer;Atherix;Megaloptera |
| Taxonomy ID: | 2078957;124299;50553 |
| Species Group: | Insect |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU000067 |
| Subject Type: | Insect |
| Subject Species: | Centroptilum triangulifer;Atherix;Megaloptera |
| Taxonomy ID: | 2078957;124299;50553 |
| Species Group: | Insect |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Heat_Treated_02 | SA002148 | FL000657 | Mayfly | species |
| Heat_Treated_02 | SA002148 | FL000657 | Mayfly Heat Treated | Treatment |
| Heat_Treated_02 | SA002148 | FL000657 | no wing pads | wing pads |
Collection:
| 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:
| Treatment ID: | TR000068 |
| Treatment Summary: | Metabolomics profile of mayfly larvae exposed to heat |
| Treatment Protocol Comments: | Heat Treated | Control | ControlX |
| Treatment: | Abiotic |
| 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:
| MB Sample ID: | SA002148 |
| 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:
| 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 |
| Apodization: | Lorentzian |
| Baseline Correction Method: | Polynomial |
| Chemical Shift Ref Std: | DSS |
