Summary of Study ST004387

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 PR002780. The data can be accessed directly via it's Project DOI: 10.21228/M89S0M This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004387
Study Title	Investigation of PPP-induced 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. We found significant effects during both the exposure and post-exposure timepoints. Acetate levels were significantly decreased at exposure in comparison to the pre-exposure timepoint in all treatments. Moreover, propionate was significantly decreased after SIVANTO prime and Cantus exposure, while the control group showed an increase in comparison to the pre-exposure timepoint. We were also able to show significant changes in correlation analyses between bacterial abundances and SCFA concentrations, which can be associated to a functional change in the bacteria related to PPP exposure.
Institute	Helmholtz Centre for Environmental Research
Department	Molecular Toxicology
Last Name	Uthoff
First Name	Cassandra
Address	Permoserstraße 15, Leipzipg, Saxony, 03418, Germany
Email	cassandra.uthoff@ufz.de
Phone	004934160252101
Submit Date	2025-11-20
Raw Data Available	Yes
Raw Data File Type(s)	mzML, wiff
Analysis Type Detail	LC-MS
Release Date	2025-12-15
Release Version	1

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Project:

Project ID:	PR002780
Project DOI:	doi: 10.21228/M89S0M
Project Title:	The structural and functional effects of plant protection products on the microbiota and host in honeybees (Apis mellifera) at environmentally relevant concentrations
Project Summary:	Plant protection products (PPPs) can reach a range of non-target organisms including pollinators such as honeybees. Even at sublethal concentrations these can influence the host physiology, development and behaviour. In this study we investigated the structural and functional effects of sublethal, environmentally relevant concentrations of PPPs on the honeybee microbiota, gut and brain, and how these potentially affect microbiota-microbiota and microbiota-host interactions. After an acute treatment we sampled an exposure timepoint three days after exposure followed by a post-exposure timepoint ten days after exposure to determine the permanence of the observed effects. Overall, this research provides novel insights into the effects of the tested products using a combination of proteomics, metabolomics, CT imaging and bioinformatic tools.
Institute:	Helmholtz Centre for Environmental Research
Department:	Molecular Toxicology
Last Name:	Engelmann
First Name:	Beatrice
Address:	Permoserstraße 15, Leipzipg, Saxony, 03418, Germany
Email:	beatrice.engelmann@ufz.de
Phone:	004934160251099

Subject:

Subject ID:	SU004546
Subject Type:	Insect
Subject Species:	Apis mellifera
Taxonomy ID:	7460

Factors:

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

mb_sample_id	local_sample_id	experimental round	treatment stage	treatment	Sample source
SA520553	E1_D3_W6_diluted	exp_round_1	exposure	Cantus	gut
SA520554	E1_D3_W1	exp_round_1	exposure	Cantus	gut
SA520555	E1_D3_W2	exp_round_1	exposure	Cantus	gut
SA520556	E1_D3_W2_diluted	exp_round_1	exposure	Cantus	gut
SA520557	E1_D3_W3	exp_round_1	exposure	Cantus	gut
SA520558	E1_D3_W3_diluted	exp_round_1	exposure	Cantus	gut
SA520559	E1_D3_W4	exp_round_1	exposure	Cantus	gut
SA520560	E1_D3_W4_diluted	exp_round_1	exposure	Cantus	gut
SA520561	E1_D3_W5	exp_round_1	exposure	Cantus	gut
SA520562	E1_D3_W5_diluted	exp_round_1	exposure	Cantus	gut
SA520563	E1_D3_W6	exp_round_1	exposure	Cantus	gut
SA520564	E1_D3_W1_diluted	exp_round_1	exposure	Cantus	gut
SA520565	E1_D3_Y1_diluted	exp_round_1	exposure	ClickPro	gut
SA520566	E1_D3_Y4_diluted	exp_round_1	exposure	ClickPro	gut
SA520567	E1_D3_Y1	exp_round_1	exposure	ClickPro	gut
SA520568	E1_D3_Y6	exp_round_1	exposure	ClickPro	gut
SA520569	E1_D3_Y5_diluted	exp_round_1	exposure	ClickPro	gut
SA520570	E1_D3_Y5	exp_round_1	exposure	ClickPro	gut
SA520571	E1_D3_Y6_diluted	exp_round_1	exposure	ClickPro	gut
SA520572	E1_D3_Y4	exp_round_1	exposure	ClickPro	gut
SA520573	E1_D3_Y3_diluted	exp_round_1	exposure	ClickPro	gut
SA520574	E1_D3_Y3	exp_round_1	exposure	ClickPro	gut
SA520575	E1_D3_Y2_diluted	exp_round_1	exposure	ClickPro	gut
SA520576	E1_D3_Y2	exp_round_1	exposure	ClickPro	gut
SA520577	E1_D3_B6	exp_round_1	exposure	Control	gut
SA520578	E1_D3_B1	exp_round_1	exposure	Control	gut
SA520579	E1_D3_B1_diluted	exp_round_1	exposure	Control	gut
SA520580	E1_D3_B2	exp_round_1	exposure	Control	gut
SA520581	E1_D3_B2_diluted	exp_round_1	exposure	Control	gut
SA520582	E1_D3_B3	exp_round_1	exposure	Control	gut
SA520583	E1_D3_B3_diluted	exp_round_1	exposure	Control	gut
SA520584	E1_D3_B4	exp_round_1	exposure	Control	gut
SA520585	E1_D3_B4_diluted	exp_round_1	exposure	Control	gut
SA520586	E1_D3_B5_diluted	exp_round_1	exposure	Control	gut
SA520587	E1_D3_B5	exp_round_1	exposure	Control	gut
SA520588	E1_D3_B6_diluted	exp_round_1	exposure	Control	gut
SA520589	E1_D3_R4_diluted	exp_round_1	exposure	SIVANTOprime	gut
SA520590	E1_D3_R1	exp_round_1	exposure	SIVANTOprime	gut
SA520591	E1_D3_R6_diluted	exp_round_1	exposure	SIVANTOprime	gut
SA520592	E1_D3_R6	exp_round_1	exposure	SIVANTOprime	gut
SA520593	E1_D3_R5_diluted	exp_round_1	exposure	SIVANTOprime	gut
SA520594	E1_D3_R5	exp_round_1	exposure	SIVANTOprime	gut
SA520595	E1_D3_R4	exp_round_1	exposure	SIVANTOprime	gut
SA520596	E1_D3_R3_diluted	exp_round_1	exposure	SIVANTOprime	gut
SA520597	E1_D3_R3	exp_round_1	exposure	SIVANTOprime	gut
SA520598	E1_D3_R2_diluted	exp_round_1	exposure	SIVANTOprime	gut
SA520599	E1_D3_R2	exp_round_1	exposure	SIVANTOprime	gut
SA520600	E1_D3_R1_diluted	exp_round_1	exposure	SIVANTOprime	gut
SA520601	E1_D10_W3	exp_round_1	post-exposure	Cantus	gut
SA520602	E1_D10_W1	exp_round_1	post-exposure	Cantus	gut
SA520603	E1_D10_W1_diluted	exp_round_1	post-exposure	Cantus	gut
SA520604	E1_D10_W2	exp_round_1	post-exposure	Cantus	gut
SA520605	E1_D10_W2_diluted	exp_round_1	post-exposure	Cantus	gut
SA520606	E1_D10_W5_diluted	exp_round_1	post-exposure	Cantus	gut
SA520607	E1_D10_W3_diluted	exp_round_1	post-exposure	Cantus	gut
SA520608	E1_D10_W4	exp_round_1	post-exposure	Cantus	gut
SA520609	E1_D10_W4_diluted	exp_round_1	post-exposure	Cantus	gut
SA520610	E1_D10_W5	exp_round_1	post-exposure	Cantus	gut
SA520611	E1_D10_W6	exp_round_1	post-exposure	Cantus	gut
SA520612	E1_D10_W6_diluted	exp_round_1	post-exposure	Cantus	gut
SA520613	E1_D10_Y2	exp_round_1	post-exposure	ClickPro	gut
SA520614	E1_D10_Y2_diluted	exp_round_1	post-exposure	ClickPro	gut
SA520615	E1_D10_Y3	exp_round_1	post-exposure	ClickPro	gut
SA520616	E1_D10_Y3_diluted	exp_round_1	post-exposure	ClickPro	gut
SA520617	E1_D10_Y1	exp_round_1	post-exposure	ClickPro	gut
SA520618	E1_D10_Y4	exp_round_1	post-exposure	ClickPro	gut
SA520619	E1_D10_Y5	exp_round_1	post-exposure	ClickPro	gut
SA520620	E1_D10_Y5_diluted	exp_round_1	post-exposure	ClickPro	gut
SA520621	E1_D10_Y6	exp_round_1	post-exposure	ClickPro	gut
SA520622	E1_D10_Y6_diluted	exp_round_1	post-exposure	ClickPro	gut
SA520623	E1_D10_Y1_diluted	exp_round_1	post-exposure	ClickPro	gut
SA520624	E1_D10_Y4_diluted	exp_round_1	post-exposure	ClickPro	gut
SA520625	E1_D10_B3	exp_round_1	post-exposure	Control	gut
SA520626	E1_D10_B1	exp_round_1	post-exposure	Control	gut
SA520627	E1_D10_B2	exp_round_1	post-exposure	Control	gut
SA520628	E1_D10_B2_diluted	exp_round_1	post-exposure	Control	gut
SA520629	E1_D10_B5	exp_round_1	post-exposure	Control	gut
SA520630	E1_D10_B3_diluted	exp_round_1	post-exposure	Control	gut
SA520631	E1_D10_B4	exp_round_1	post-exposure	Control	gut
SA520632	E1_D10_B4_diluted	exp_round_1	post-exposure	Control	gut
SA520633	E1_D10_B5_diluted	exp_round_1	post-exposure	Control	gut
SA520634	E1_D10_B6	exp_round_1	post-exposure	Control	gut
SA520635	E1_D10_B6_diluted	exp_round_1	post-exposure	Control	gut
SA520636	E1_D10_B1_diluted	exp_round_1	post-exposure	Control	gut
SA520637	E1_D10_R4_diluted	exp_round_1	post-exposure	SIVANTOprime	gut
SA520638	E1_D10_R6_diluted	exp_round_1	post-exposure	SIVANTOprime	gut
SA520639	E1_D10_R2_diluted	exp_round_1	post-exposure	SIVANTOprime	gut
SA520640	E1_D10_R6	exp_round_1	post-exposure	SIVANTOprime	gut
SA520641	E1_D10_R1	exp_round_1	post-exposure	SIVANTOprime	gut
SA520642	E1_D10_R2	exp_round_1	post-exposure	SIVANTOprime	gut
SA520643	E1_D10_R1_diluted	exp_round_1	post-exposure	SIVANTOprime	gut
SA520644	E1_D10_R3	exp_round_1	post-exposure	SIVANTOprime	gut
SA520645	E1_D10_R4	exp_round_1	post-exposure	SIVANTOprime	gut
SA520646	E1_D10_R5	exp_round_1	post-exposure	SIVANTOprime	gut
SA520647	E1_D10_R3_diluted	exp_round_1	post-exposure	SIVANTOprime	gut
SA520648	E1_D10_R5_diluted	exp_round_1	post-exposure	SIVANTOprime	gut
SA520649	E1_D0_W4	exp_round_1	pre_exposure	Cantus	gut
SA520650	E1_D0_W5_diluted	exp_round_1	pre_exposure	Cantus	gut
SA520651	E1_D0_W5	exp_round_1	pre_exposure	Cantus	gut
SA520652	E1_D0_W4_diluted	exp_round_1	pre_exposure	Cantus	gut

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Collection:

Collection ID:	CO004539
Collection Summary:	Workers were pooled to obtain a final sample number of n = 6 per treatment per timepoint per experimental round. We pooled three guts per sample. In total, across the three experimental rounds, this resulted in 18 samples from each treatment and each timepoint. 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:	TR004555
Treatment Summary:	In brief, four Dadant US observation hives were housed in a temperature-controlled, dark room with access to the outside via an east-facing tube. Colonies were established seven to ten days before the start of each experimental round and contained egg-laying sister queens. Treatments were assigned randomly to hives in each experimental round to avoid any hive or location effects. Newly emerged workers were reared in an incubator and randomly assigned to one treatment group. They were marked on the thorax, using the different colours assigned to the PPPs to represent hive origin and treatment (n=260-300 per treatment). PPPs exposure began three days after the newly emerged workers were placed into their hives. The PPPs at their concentrations described in section 2.2 were mixed into a 50% (w/v) sucrose solution (control hives received only sucrose solution). The bees were treated once with one liter of treatment solution, adapted from the protocols presented by Medrzycki et al. (2013). Honeybee sampling started at the pre-exposure timepoint just before PPPs were fed to the colonies, when the marked workers were three days old. The second timepoint was three days afterwards and was considered the exposure timepoint. The third and last time point was the post-exposure timepoint, where samples were taken ten days after the pre-exposure (seven days after exposure) timepoint.

Treatment ID:

TR004555

Treatment Summary:

In brief, four Dadant US observation hives were housed in a temperature-controlled, dark room with access to the outside via an east-facing tube. Colonies were established seven to ten days before the start of each experimental round and contained egg-laying sister queens. Treatments were assigned randomly to hives in each experimental round to avoid any hive or location effects. Newly emerged workers were reared in an incubator and randomly assigned to one treatment group. They were marked on the thorax, using the different colours assigned to the PPPs to represent hive origin and treatment (n=260-300 per treatment). PPPs exposure began three days after the newly emerged workers were placed into their hives. The PPPs at their concentrations described in section 2.2 were mixed into a 50% (w/v) sucrose solution (control hives received only sucrose solution). The bees were treated once with one liter of treatment solution, adapted from the protocols presented by Medrzycki et al. (2013). Honeybee sampling started at the pre-exposure timepoint just before PPPs were fed to the colonies, when the marked workers were three days old. The second timepoint was three days afterwards and was considered the exposure timepoint. The third and last time point was the post-exposure timepoint, where samples were taken ten days after the pre-exposure (seven days after exposure) timepoint.

Sample Preparation:

Sampleprep ID:	SP004552
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:

SP004552

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	AN007330
Chromatography ID	CH005562
MS ID	MS007024
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000 RS
Column	Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 µm)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex 5500 QTrap
Ion Mode	NEGATIVE
Units	µMol

Chromatography:

Chromatography ID:	CH005562
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 µm)
Column Temperature:	40°C
Flow Gradient:	0-2 min: 15% B, 2-17 min: 15-50% B, 17-17.1 min: 50-100% B, 17.1-18 min: 100% B, 18-18.1 min: 100-15% B, 18.1-21 min: 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:	MS007024
Analysis ID:	AN007330
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