Summary of Study ST003554

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002188. The data can be accessed directly via it's Project DOI: 10.21228/M8SV5J This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003554
Study Title	Investigation of age-dependent changes in the brain of honeybee workers using targeted metabolomics for amino acid and biogenic amine determination
Study Summary	In the honeybee guts we measured the concentrations of 21 amino acids and 6 biogenic amines. Using a k-means clustering, the metabolites were grouped by their trends into six clusters. Tyramine and tyrosine are involved in the octopamine and dopamine biosynthesis. We found that all four metabolites were part of the same cluster, with their concentrations being significantly decreased in comparison to newly emerged workers from day 1 onwards for the entire duration of the study.
Institute	Helmholtz Centre for Environmental Research
Department	Molecular Systems Biology
Last Name	Engelmann
First Name	Beatrice
Address	Permoserstraße 15, Leipzipg, Saxony, 03418, Germany
Email	beatrice.engelmann@ufz.de
Phone	004934160251099
Submit Date	2024-10-28
Raw Data Available	Yes
Raw Data File Type(s)	wiff
Analysis Type Detail	LC-MS
Release Date	2024-11-27
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002188
Project DOI:	doi: 10.21228/M8SV5J
Project Title:	Comprehension of the age-dependent gut and brain interaction of honeybee workers by integration of multi omics approaches
Project Summary:	In honeybees, division of labour is a key feature, with age-related behavioural transitions being closely associated with molecular changes in the brain, gut, and microbiota. In this study, to investigate these molecular changes and thus better understand their contribution to behavioural responses and modulation, we analysed the global metabolomic shifts in honeybee workers and their microbiota throughout their lives. Overall, our findings provide new insights toward developing potential biomarkers for evaluation of different functional changes related to various environmental stressors.
Institute:	Helmholtz Centre for Environmental Research
Department:	Molecular Systems Biology
Last Name:	Engelmann
First Name:	Beatrice
Address:	Permoserstraße 15, Leipzipg, Saxony, 03418, Germany
Email:	beatrice.engelmann@ufz.de
Phone:	004934160251099

Subject:

Subject ID:	SU003683
Subject Type:	Insect
Subject Species:	Apis mellifera
Taxonomy ID:	7460
Species Group:	Insects

Factors:

Subject type: Insect; Subject species: Apis mellifera (Factor headings shown in green)

mb_sample_id	local_sample_id	life stage	age of workers in days	Sample source
SA388307	H2_D1_3_diluted	establishment	1	brain
SA388308	H1_D1_3_diluted	establishment	1	brain
SA388309	H1_D1_4_diluted	establishment	1	brain
SA388310	H1_D1_5_diluted	establishment	1	brain
SA388311	H2_D1_1	establishment	1	brain
SA388312	H2_D1_2	establishment	1	brain
SA388313	H2_D1_3	establishment	1	brain
SA388314	H2_D1_4	establishment	1	brain
SA388315	H2_D1_5	establishment	1	brain
SA388316	H2_D1_1_diluted	establishment	1	brain
SA388317	H2_D1_2_diluted	establishment	1	brain
SA388318	H2_D1_5_diluted	establishment	1	brain
SA388319	H1_D1_1_diluted	establishment	1	brain
SA388320	H3_D1_1	establishment	1	brain
SA388321	H3_D1_2	establishment	1	brain
SA388322	H3_D1_3	establishment	1	brain
SA388323	H3_D1_4	establishment	1	brain
SA388324	H3_D1_5	establishment	1	brain
SA388325	H3_D1_1_diluted	establishment	1	brain
SA388326	H3_D1_2_diluted	establishment	1	brain
SA388327	H3_D1_3_diluted	establishment	1	brain
SA388328	H3_D1_4_diluted	establishment	1	brain
SA388329	H3_D1_5_diluted	establishment	1	brain
SA388330	H1_D1_2_diluted	establishment	1	brain
SA388331	H2_D1_4_diluted	establishment	1	brain
SA388332	H1_D1_5	establishment	1	brain
SA388333	H1_D1_4	establishment	1	brain
SA388334	H1_D1_3	establishment	1	brain
SA388335	H1_D1_1	establishment	1	brain
SA388336	H1_D1_2	establishment	1	brain
SA388337	H2_D3_3	establishment	3	brain
SA388338	H2_D3_2	establishment	3	brain
SA388339	H2_D3_1	establishment	3	brain
SA388340	H2_D3_3_diluted	establishment	3	brain
SA388341	H3_D3_1_diluted	establishment	3	brain
SA388342	H2_D3_4	establishment	3	brain
SA388343	H2_D3_5	establishment	3	brain
SA388344	H3_D3_2_diluted	establishment	3	brain
SA388345	H3_D3_3_diluted	establishment	3	brain
SA388346	H3_D3_4_diluted	establishment	3	brain
SA388347	H3_D3_5_diluted	establishment	3	brain
SA388348	H1_D3_2	establishment	3	brain
SA388349	H1_D3_1	establishment	3	brain
SA388350	H2_D3_2_diluted	establishment	3	brain
SA388351	H2_D3_5_diluted	establishment	3	brain
SA388352	H1_D3_2_diluted	establishment	3	brain
SA388353	H2_D3_1_diluted	establishment	3	brain
SA388354	H1_D3_3	establishment	3	brain
SA388355	H2_D3_4_diluted	establishment	3	brain
SA388356	H3_D3_5	establishment	3	brain
SA388357	H3_D3_4	establishment	3	brain
SA388358	H3_D3_3	establishment	3	brain
SA388359	H3_D3_2	establishment	3	brain
SA388360	H3_D3_1	establishment	3	brain
SA388361	H1_D3_1_diluted	establishment	3	brain
SA388362	H1_D3_3_diluted	establishment	3	brain
SA388363	H1_D3_4_diluted	establishment	3	brain
SA388364	H1_D3_5_diluted	establishment	3	brain
SA388365	H1_D3_4	establishment	3	brain
SA388366	H1_D3_5	establishment	3	brain
SA388367	H2_D5_1_diluted	establishment	5	brain
SA388368	H2_D5_5_diluted	establishment	5	brain
SA388369	H3_D5_5	establishment	5	brain
SA388370	H2_D5_4_diluted	establishment	5	brain
SA388371	H2_D5_3_diluted	establishment	5	brain
SA388372	H2_D5_2_diluted	establishment	5	brain
SA388373	H2_D5_4	establishment	5	brain
SA388374	H1_D5_1	establishment	5	brain
SA388375	H2_D5_5	establishment	5	brain
SA388376	H3_D5_4	establishment	5	brain
SA388377	H2_D5_3	establishment	5	brain
SA388378	H3_D5_1_diluted	establishment	5	brain
SA388379	H3_D5_3	establishment	5	brain
SA388380	H3_D5_2	establishment	5	brain
SA388381	H3_D5_1	establishment	5	brain
SA388382	H1_D5_1_diluted	establishment	5	brain
SA388383	H1_D5_2_diluted	establishment	5	brain
SA388384	H1_D5_3_diluted	establishment	5	brain
SA388385	H1_D5_4_diluted	establishment	5	brain
SA388386	H1_D5_5_diluted	establishment	5	brain
SA388387	H3_D5_5_diluted	establishment	5	brain
SA388388	H3_D5_4_diluted	establishment	5	brain
SA388389	H3_D5_3_diluted	establishment	5	brain
SA388390	H3_D5_2_diluted	establishment	5	brain
SA388391	H1_D5_2	establishment	5	brain
SA388392	H2_D5_2	establishment	5	brain
SA388393	H1_D5_5	establishment	5	brain
SA388394	H1_D5_4	establishment	5	brain
SA388395	H1_D5_3	establishment	5	brain
SA388396	H2_D5_1	establishment	5	brain
SA388397	H3_EF_4	experienced_forager	>12	brain
SA388398	H3_EF_3	experienced_forager	>12	brain
SA388399	H3_EF_2	experienced_forager	>12	brain
SA388400	H3_EF_1	experienced_forager	>12	brain
SA388401	H2_EF_1_diluted	experienced_forager	>12	brain
SA388402	H2_EF_2_diluted	experienced_forager	>12	brain
SA388403	H2_EF_3_diluted	experienced_forager	>12	brain
SA388404	H2_EF_4_diluted	experienced_forager	>12	brain
SA388405	H2_EF_5_diluted	experienced_forager	>12	brain
SA388406	H3_EF_5	experienced_forager	>12	brain

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Collection:

Collection ID:	CO003676
Collection Summary:	Workers were pooled to obtain a final sample number of n = 5. The brain(s) and gut(s) from the same workers were assigned the same sample names. In total, we had 15 samples from each age group, with the exception of the foraging stages, where owing to the lack of foragers from Hive 1, we only had 11. All sampled bees were placed directly in liquid nitrogen to prevent changes in the metabolic profile. They were then decapitated in the laboratory so that the head and body of each bee were kept in a separate Eppendorf tube (Eppendorf, Germany) for later dissection. Honeybee brains were dissected using a dissecting microscope (SZX7, Olympus). The head was placed on a petri dish lined with SYLGARD® 184 silicone. Before pinning the head for dissection, antennae and mouthpieces were removed. Subsequently, double-distilled water (ddH2O) was added until the head was fully submerged to facilitate dissection. The head cuticle was removed, and the hypopharyngeal glands and trachea were extracted from the inside. The brain was then separated from the cuticle and remaining tracheal remnants were removed before transferring the clean brain to a new Eppendorf tube.
Sample Type:	Bee brain

Treatment:

Treatment ID:	TR003692
Treatment Summary:	On the day of newly emerged workers (day 0), bees were samples as a baseline for no or low bacterial abundance in the gut. Moreover, bees from each hive were collected on Day 1, 3 and 5 to cover the establishment phase of the gut. Samples for the in-hive worker phase, defined as workers inside the hives that have a fully developed microbiome, were taken every day from Day 7 to Day 10. For the foraging phase, we differentiated between new and experienced foragers, as these show different behaviour due to different levels of experience.Based on experience, they were either collected one to two days after onset (new foragers) or at least four days after onset (experienced foragers).

Treatment ID:

TR003692

Treatment Summary:

On the day of newly emerged workers (day 0), bees were samples as a baseline for no or low bacterial abundance in the gut. Moreover, bees from each hive were collected on Day 1, 3 and 5 to cover the establishment phase of the gut. Samples for the in-hive worker phase, defined as workers inside the hives that have a fully developed microbiome, were taken every day from Day 7 to Day 10. For the foraging phase, we differentiated between new and experienced foragers, as these show different behaviour due to different levels of experience.Based on experience, they were either collected one to two days after onset (new foragers) or at least four days after onset (experienced foragers).

Sample Preparation:

Sampleprep ID:	SP003690
Sampleprep Summary:	In brief, x mg pool brain was mixed with five times the volume (in µL) of acetonitrile (ACN):Water (1:1, v/v) and homogenized using a TissueLyser II (30 Hz, 10 min; Retsch Qiagen). After Centrifugation (2 min, 14000 rpm), 10 µL were used for amino acid derivatization. First, the supernatant was evaporated to dryness (SpeedVac, Eppendorf), resuspended in 50 µL of 5% phenyl isothiocyanate (PITC) in ethanol: H2O: pyridine (1:1:1, v/v/v), and incubated for 25 min at RT. Subsequently, the samples were dried to remove excess PITC and resuspended in 10 µL 5 mM ammonium acetate in methanol. After incubation (10 min at 14,000 rpm) 90 µL of H2O: ACN + 0.2% formic acid were added. Each derivative was measured in an undiluted and diluted (1:25) manner.

Sampleprep ID:

SP003690

Sampleprep Summary:

In brief, x mg pool brain was mixed with five times the volume (in µL) of acetonitrile (ACN):Water (1:1, v/v) and homogenized using a TissueLyser II (30 Hz, 10 min; Retsch Qiagen). After Centrifugation (2 min, 14000 rpm), 10 µL were used for amino acid derivatization. First, the supernatant was evaporated to dryness (SpeedVac, Eppendorf), resuspended in 50 µL of 5% phenyl isothiocyanate (PITC) in ethanol: H2O: pyridine (1:1:1, v/v/v), and incubated for 25 min at RT. Subsequently, the samples were dried to remove excess PITC and resuspended in 10 µL 5 mM ammonium acetate in methanol. After incubation (10 min at 14,000 rpm) 90 µL of H2O: ACN + 0.2% formic acid were added. Each derivative was measured in an undiluted and diluted (1:25) manner.

Combined analysis:

Analysis ID	AN005842	AN005843
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Waters Acquity	Waters Acquity
Column	Zorbax Eclipse XDB-C18 (100 x 3.0mm, 3.5um)	Zorbax Eclipse XDB-C18 (100 x 3.0mm, 3.5um)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	ABI Sciex 5500 QTrap	ABI Sciex 6500+ Qtrap
Ion Mode	POSITIVE	POSITIVE
Units	µMol	µMol

Chromatography:

Chromatography ID:	CH004437
Instrument Name:	Waters Acquity
Column Name:	Zorbax Eclipse XDB-C18 (100 x 3.0mm, 3.5um)
Column Temperature:	50
Flow Gradient:	0-0.5 min at 0% B, 0.5-4 min 0-70% B, 4-5.3 min 70% B, 5.3-5.4 min 70-0% B, 5.4-7.3 min 0% B
Flow Rate:	0.5 mL/min
Solvent A:	100% water; 0.2% formic acid
Solvent B:	100% acetonitrile; 0.2% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005562
Analysis ID:	AN005842
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	For identification and quantitation, a scheduled MRM method was used, with specific transitions for every amino acid and biogenic amine. Data acquisition and peak integration were performed in SciexOS software (Version 3.0.0.). Calculation of concentration was done using external calibration curves.
Ion Mode:	POSITIVE

MS ID:	MS005563
Analysis ID:	AN005843
Instrument Name:	ABI Sciex 6500+ Qtrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	For identification and quantitation, a scheduled MRM method was used, with specific transitions for every amino acid and biogenic amine. Data acquisition and peak integration were performed in SciexOS software (Version 3.0.0.). Calculation of concentration was done using external calibration curves.
Ion Mode:	POSITIVE