Summary of Study ST003552

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	ST003552
Study Title	Investigation of age-dependent changes in the gut of honeybee workers using targeted metabolomics for SCFA determination
Study Summary	To determine changes in the metabolic activity of the gut microbiota, we measured the concentrations of seven short-chain fatty acids (SCFA) and lactate. All SCFA showed low concentrations in newly emerged workers. We found that acetate, propionate and butyrate are the main SCFA in the honeybee gut and peak in 5-day old and in-hive workers. Lactate showed an inverse trend with highest concentrations in 1-day old workers and a significant decrease on all subsequent days (Kruskal Wallis test, p <0.05).
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	2024-10-21
Raw Data Available	Yes
Raw Data File Type(s)	wiff
Analysis Type Detail	LC-MS
Release Date	2024-11-27
Release Version	1

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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:	SU003681
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
SA387919	H2_D1_4_diluted	establishment	1	gut
SA387920	H2_D1_4	establishment	1	gut
SA387921	H2_D1_5	establishment	1	gut
SA387922	H3_D1_1	establishment	1	gut
SA387923	H3_D1_2	establishment	1	gut
SA387924	H3_D1_3	establishment	1	gut
SA387925	H3_D1_4	establishment	1	gut
SA387926	H3_D1_5	establishment	1	gut
SA387927	H2_D1_3_diluted	establishment	1	gut
SA387928	H2_D1_1	establishment	1	gut
SA387929	H2_D1_2_diluted	establishment	1	gut
SA387930	H2_D1_1_diluted	establishment	1	gut
SA387931	H1_D1_5_diluted	establishment	1	gut
SA387932	H1_D1_4_diluted	establishment	1	gut
SA387933	H1_D1_3_diluted	establishment	1	gut
SA387934	H1_D1_2_diluted	establishment	1	gut
SA387935	H1_D1_1_diluted	establishment	1	gut
SA387936	H2_D1_2	establishment	1	gut
SA387937	H2_D1_3	establishment	1	gut
SA387938	H1_D1_5	establishment	1	gut
SA387939	H3_D1_1_diluted	establishment	1	gut
SA387940	H1_D1_4	establishment	1	gut
SA387941	H3_D1_5_diluted	establishment	1	gut
SA387942	H3_D1_4_diluted	establishment	1	gut
SA387943	H3_D1_3_diluted	establishment	1	gut
SA387944	H3_D1_2_diluted	establishment	1	gut
SA387945	H2_D1_5_diluted	establishment	1	gut
SA387946	H1_D1_1	establishment	1	gut
SA387947	H1_D1_2	establishment	1	gut
SA387948	H1_D1_3	establishment	1	gut
SA387949	H2_D3_2_diluted	establishment	3	gut
SA387950	H3_D3_4_diluted	establishment	3	gut
SA387951	H3_D3_3_diluted	establishment	3	gut
SA387952	H3_D3_2_diluted	establishment	3	gut
SA387953	H3_D3_1_diluted	establishment	3	gut
SA387954	H2_D3_5_diluted	establishment	3	gut
SA387955	H1_D3_1_diluted	establishment	3	gut
SA387956	H2_D3_1_diluted	establishment	3	gut
SA387957	H1_D3_5_diluted	establishment	3	gut
SA387958	H1_D3_4_diluted	establishment	3	gut
SA387959	H1_D3_3_diluted	establishment	3	gut
SA387960	H1_D3_2_diluted	establishment	3	gut
SA387961	H2_D3_4_diluted	establishment	3	gut
SA387962	H3_D3_5_diluted	establishment	3	gut
SA387963	H2_D3_3_diluted	establishment	3	gut
SA387964	H3_D3_2	establishment	3	gut
SA387965	H2_D3_5	establishment	3	gut
SA387966	H1_D3_1	establishment	3	gut
SA387967	H1_D3_2	establishment	3	gut
SA387968	H1_D3_3	establishment	3	gut
SA387969	H1_D3_4	establishment	3	gut
SA387970	H1_D3_5	establishment	3	gut
SA387971	H2_D3_1	establishment	3	gut
SA387972	H2_D3_2	establishment	3	gut
SA387973	H3_D3_5	establishment	3	gut
SA387974	H3_D3_4	establishment	3	gut
SA387975	H3_D3_3	establishment	3	gut
SA387976	H2_D3_3	establishment	3	gut
SA387977	H2_D3_4	establishment	3	gut
SA387978	H3_D3_1	establishment	3	gut
SA387979	H1_D5_2	establishment	5	gut
SA387980	H3_D5_1_diluted	establishment	5	gut
SA387981	H1_D5_3	establishment	5	gut
SA387982	H1_D5_1_diluted	establishment	5	gut
SA387983	H1_D5_2_diluted	establishment	5	gut
SA387984	H1_D5_3_diluted	establishment	5	gut
SA387985	H2_D5_4_diluted	establishment	5	gut
SA387986	H2_D5_3_diluted	establishment	5	gut
SA387987	H2_D5_2_diluted	establishment	5	gut
SA387988	H2_D5_1_diluted	establishment	5	gut
SA387989	H1_D5_4_diluted	establishment	5	gut
SA387990	H2_D5_5_diluted	establishment	5	gut
SA387991	H3_D5_2_diluted	establishment	5	gut
SA387992	H3_D5_5	establishment	5	gut
SA387993	H1_D5_4	establishment	5	gut
SA387994	H1_D5_5	establishment	5	gut
SA387995	H2_D5_1	establishment	5	gut
SA387996	H2_D5_2	establishment	5	gut
SA387997	H2_D5_3	establishment	5	gut
SA387998	H2_D5_4	establishment	5	gut
SA387999	H2_D5_5	establishment	5	gut
SA388000	H3_D5_1	establishment	5	gut
SA388001	H3_D5_2	establishment	5	gut
SA388002	H3_D5_3	establishment	5	gut
SA388003	H3_D5_4	establishment	5	gut
SA388004	H1_D5_5_diluted	establishment	5	gut
SA388005	H3_D5_3_diluted	establishment	5	gut
SA388006	H1_D5_1	establishment	5	gut
SA388007	H3_D5_5_diluted	establishment	5	gut
SA388008	H3_D5_4_diluted	establishment	5	gut
SA388009	H3_EF_5_diluted	experienced_forager	>12	gut
SA388010	H3_EF_3_diluted	experienced_forager	>12	gut
SA388011	H3_EF_2_diluted	experienced_forager	>12	gut
SA388012	H3_EF_1_diluted	experienced_forager	>12	gut
SA388013	H2_EF_5_diluted	experienced_forager	>12	gut
SA388014	H2_EF_4_diluted	experienced_forager	>12	gut
SA388015	H2_EF_3_diluted	experienced_forager	>12	gut
SA388016	H1_EF_1_diluted	experienced_forager	>12	gut
SA388017	H2_EF_1_diluted	experienced_forager	>12	gut
SA388018	H2_EF_2_diluted	experienced_forager	>12	gut

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Collection:

Collection ID:	CO003674
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. Worker guts were dissected on Petri dishes. Sternite 6 and the stinger apparatus were gently pulled using tweezers, which allowed extraction of the entire gastrointestinal tract. The stinger apparatus and venom sac were carefully excised and the honey crop was removed.
Sample Type:	Bee gut

Treatment:

Treatment ID:	TR003690
Treatment Summary:	On the day of newly emerged workers (day 0), bees were sampled 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:

TR003690

Treatment Summary:

On the day of newly emerged workers (day 0), bees were sampled 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:	SP003688
Sampleprep Summary:	In brief, x mg pool gut 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) 40 µl were used for further derivatization of short chain fatty acids. Each sample was mixed with ACN to a final concentration of 50 %. SCFAs were derivatized with 200 mM 3-nitrophenylhydrazine and 120 mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride in pyridine and then diluted in 10 % ACN.

Sampleprep ID:

SP003688

Sampleprep Summary:

In brief, x mg pool gut 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) 40 µl were used for further derivatization of short chain fatty acids. Each sample was mixed with ACN to a final concentration of 50 %. SCFAs were derivatized with 200 mM 3-nitrophenylhydrazine and 120 mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride in pyridine and then diluted in 10 % ACN.

Combined analysis:

Analysis ID	AN005839
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000 RS
Column	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex 5500 QTrap
Ion Mode	NEGATIVE
Units	µMol

Chromatography:

Chromatography ID:	CH004435
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	40
Flow Gradient:	0-2min: 15% B, 2-17min: 15-50% B, 17-17.1min: 50-100% B, 17.1-18min: 100% B, 18-18.1min: 100-15% B, 18.1-21min: 15% B
Flow Rate:	0.35 mL/min
Solvent A:	100% water; 0.01% formic acid
Solvent B:	100% acetonitrile; 0.01% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005559
Analysis ID:	AN005839
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 SCFA. Data acquisition and peak integration were performed in SciexOS software (Version 3.0.0.). Calculation of concentration was done using external calibration curves and statistics were performed using the R software programme.
Ion Mode:	NEGATIVE