Summary of Study ST004408

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	ST004408
Study Title	Investigation of PPP induced changes in the gut of honeybee workers using targeted metabolomics for amino acid and biogenic amine determination.
Study Summary	In the honeybee guts we measured the concentrations of 20 amino acids and 6 biogenic amines. Ornithine was significantly more decreased after Cantus and ClickPro exposure in comparison the control at both time points, and the same was observed for dopamine at the exposure timepoint. Meanwhile, octopamine was significantly increased in the Cantus group at the post-exposure timepoint, while the control bees showed a decrease in concentration in comparison to the pre-exposure timepoint.
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	2025-12-22
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:	SU004567
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
SA521441	E1_D3_W6_diluted	exp_round_1	exposure	Cantus	Bee gut
SA521442	E1_D3_W1	exp_round_1	exposure	Cantus	Bee gut
SA521443	E1_D3_W2	exp_round_1	exposure	Cantus	Bee gut
SA521444	E1_D3_W2_diluted	exp_round_1	exposure	Cantus	Bee gut
SA521445	E1_D3_W3	exp_round_1	exposure	Cantus	Bee gut
SA521446	E1_D3_W3_diluted	exp_round_1	exposure	Cantus	Bee gut
SA521447	E1_D3_W4	exp_round_1	exposure	Cantus	Bee gut
SA521448	E1_D3_W4_diluted	exp_round_1	exposure	Cantus	Bee gut
SA521449	E1_D3_W5	exp_round_1	exposure	Cantus	Bee gut
SA521450	E1_D3_W5_diluted	exp_round_1	exposure	Cantus	Bee gut
SA521451	E1_D3_W6	exp_round_1	exposure	Cantus	Bee gut
SA521452	E1_D3_W1_diluted	exp_round_1	exposure	Cantus	Bee gut
SA521453	E1_D3_Y1_diluted	exp_round_1	exposure	ClickPro	Bee gut
SA521454	E1_D3_Y4_diluted	exp_round_1	exposure	ClickPro	Bee gut
SA521455	E1_D3_Y1	exp_round_1	exposure	ClickPro	Bee gut
SA521456	E1_D3_Y6	exp_round_1	exposure	ClickPro	Bee gut
SA521457	E1_D3_Y5_diluted	exp_round_1	exposure	ClickPro	Bee gut
SA521458	E1_D3_Y5	exp_round_1	exposure	ClickPro	Bee gut
SA521459	E1_D3_Y6_diluted	exp_round_1	exposure	ClickPro	Bee gut
SA521460	E1_D3_Y4	exp_round_1	exposure	ClickPro	Bee gut
SA521461	E1_D3_Y3_diluted	exp_round_1	exposure	ClickPro	Bee gut
SA521462	E1_D3_Y3	exp_round_1	exposure	ClickPro	Bee gut
SA521463	E1_D3_Y2_diluted	exp_round_1	exposure	ClickPro	Bee gut
SA521464	E1_D3_Y2	exp_round_1	exposure	ClickPro	Bee gut
SA521465	E1_D3_B6	exp_round_1	exposure	Control	Bee gut
SA521466	E1_D3_B1	exp_round_1	exposure	Control	Bee gut
SA521467	E1_D3_B1_diluted	exp_round_1	exposure	Control	Bee gut
SA521468	E1_D3_B2	exp_round_1	exposure	Control	Bee gut
SA521469	E1_D3_B2_diluted	exp_round_1	exposure	Control	Bee gut
SA521470	E1_D3_B3	exp_round_1	exposure	Control	Bee gut
SA521471	E1_D3_B3_diluted	exp_round_1	exposure	Control	Bee gut
SA521472	E1_D3_B4	exp_round_1	exposure	Control	Bee gut
SA521473	E1_D3_B4_diluted	exp_round_1	exposure	Control	Bee gut
SA521474	E1_D3_B5_diluted	exp_round_1	exposure	Control	Bee gut
SA521475	E1_D3_B5	exp_round_1	exposure	Control	Bee gut
SA521476	E1_D3_B6_diluted	exp_round_1	exposure	Control	Bee gut
SA521477	E1_D3_R4_diluted	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521478	E1_D3_R1	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521479	E1_D3_R6_diluted	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521480	E1_D3_R6	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521481	E1_D3_R5_diluted	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521482	E1_D3_R5	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521483	E1_D3_R4	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521484	E1_D3_R3_diluted	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521485	E1_D3_R3	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521486	E1_D3_R2_diluted	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521487	E1_D3_R2	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521488	E1_D3_R1_diluted	exp_round_1	exposure	SIVANTOprime	Bee gut
SA521489	E1_D10_W3	exp_round_1	post-exposure	Cantus	Bee gut
SA521490	E1_D10_W1	exp_round_1	post-exposure	Cantus	Bee gut
SA521491	E1_D10_W1_diluted	exp_round_1	post-exposure	Cantus	Bee gut
SA521492	E1_D10_W2	exp_round_1	post-exposure	Cantus	Bee gut
SA521493	E1_D10_W2_diluted	exp_round_1	post-exposure	Cantus	Bee gut
SA521494	E1_D10_W5_diluted	exp_round_1	post-exposure	Cantus	Bee gut
SA521495	E1_D10_W3_diluted	exp_round_1	post-exposure	Cantus	Bee gut
SA521496	E1_D10_W4	exp_round_1	post-exposure	Cantus	Bee gut
SA521497	E1_D10_W4_diluted	exp_round_1	post-exposure	Cantus	Bee gut
SA521498	E1_D10_W5	exp_round_1	post-exposure	Cantus	Bee gut
SA521499	E1_D10_W6	exp_round_1	post-exposure	Cantus	Bee gut
SA521500	E1_D10_W6_diluted	exp_round_1	post-exposure	Cantus	Bee gut
SA521501	E1_D10_Y2	exp_round_1	post-exposure	ClickPro	Bee gut
SA521502	E1_D10_Y2_diluted	exp_round_1	post-exposure	ClickPro	Bee gut
SA521503	E1_D10_Y3	exp_round_1	post-exposure	ClickPro	Bee gut
SA521504	E1_D10_Y3_diluted	exp_round_1	post-exposure	ClickPro	Bee gut
SA521505	E1_D10_Y1	exp_round_1	post-exposure	ClickPro	Bee gut
SA521506	E1_D10_Y4	exp_round_1	post-exposure	ClickPro	Bee gut
SA521507	E1_D10_Y5	exp_round_1	post-exposure	ClickPro	Bee gut
SA521508	E1_D10_Y5_diluted	exp_round_1	post-exposure	ClickPro	Bee gut
SA521509	E1_D10_Y6	exp_round_1	post-exposure	ClickPro	Bee gut
SA521510	E1_D10_Y6_diluted	exp_round_1	post-exposure	ClickPro	Bee gut
SA521511	E1_D10_Y1_diluted	exp_round_1	post-exposure	ClickPro	Bee gut
SA521512	E1_D10_Y4_diluted	exp_round_1	post-exposure	ClickPro	Bee gut
SA521513	E1_D10_B3	exp_round_1	post-exposure	Control	Bee gut
SA521514	E1_D10_B1	exp_round_1	post-exposure	Control	Bee gut
SA521515	E1_D10_B2	exp_round_1	post-exposure	Control	Bee gut
SA521516	E1_D10_B2_diluted	exp_round_1	post-exposure	Control	Bee gut
SA521517	E1_D10_B5	exp_round_1	post-exposure	Control	Bee gut
SA521518	E1_D10_B3_diluted	exp_round_1	post-exposure	Control	Bee gut
SA521519	E1_D10_B4	exp_round_1	post-exposure	Control	Bee gut
SA521520	E1_D10_B4_diluted	exp_round_1	post-exposure	Control	Bee gut
SA521521	E1_D10_B5_diluted	exp_round_1	post-exposure	Control	Bee gut
SA521522	E1_D10_B6	exp_round_1	post-exposure	Control	Bee gut
SA521523	E1_D10_B6_diluted	exp_round_1	post-exposure	Control	Bee gut
SA521524	E1_D10_B1_diluted	exp_round_1	post-exposure	Control	Bee gut
SA521525	E1_D10_R4_diluted	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521526	E1_D10_R6_diluted	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521527	E1_D10_R2_diluted	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521528	E1_D10_R6	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521529	E1_D10_R1	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521530	E1_D10_R2	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521531	E1_D10_R1_diluted	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521532	E1_D10_R3	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521533	E1_D10_R4	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521534	E1_D10_R5	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521535	E1_D10_R3_diluted	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521536	E1_D10_R5_diluted	exp_round_1	post-exposure	SIVANTOprime	Bee gut
SA521537	E1_D0_W4	exp_round_1	pre_exposure	Cantus	Bee gut
SA521538	E1_D0_W5_diluted	exp_round_1	pre_exposure	Cantus	Bee gut
SA521539	E1_D0_W5	exp_round_1	pre_exposure	Cantus	Bee gut
SA521540	E1_D0_W4_diluted	exp_round_1	pre_exposure	Cantus	Bee gut

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Collection:

Collection ID:	CO004560
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:	TR004576
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 litre 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:

TR004576

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 litre 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:	SP004573
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), 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:

SP004573

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), 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	AN007371	AN007372
Chromatography ID	CH005584	CH005584
MS ID	MS007065	MS007066
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Waters Acquity	Waters Acquity
Column	Zorbax Eclipse XDB-C18 (100 x 3.0 mm, 3.5 µm)	Zorbax Eclipse XDB-C18 (100 x 3.0 mm, 3.5 µm)
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:	CH005584
Instrument Name:	Waters Acquity
Column Name:	Zorbax Eclipse XDB-C18 (100 x 3.0 mm, 3.5 µm)
Column Temperature:	50°C
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:	MS007065
Analysis ID:	AN007371
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:	MS007066
Analysis ID:	AN007372
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