Summary of Study ST004428

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	ST004428
Study Title	Investigation of PPP-induced 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. Four out of the 21 amino acids showed significant changes during the exposure phase, while three were significantly affected at post-exposure. Out of these, only threonine was significantly affected at both exposure and post-exposure and showed a significant decrease after Cantus exposure. Out of the biogenic amines, only tyramine was affected during the exposure timepoint and showed significantly less of a decrease in all treatments in comparison to the control.
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	2025-12-29
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:	SU004588
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
SA522798	E1_D3_W5	exp_round_1	exposure	Cantus	Bee brain
SA522799	E1_D3_W1	exp_round_1	exposure	Cantus	Bee brain
SA522800	E1_D3_W2	exp_round_1	exposure	Cantus	Bee brain
SA522801	E1_D3_W7_diluted	exp_round_1	exposure	Cantus	Bee brain
SA522802	E1_D3_W6_diluted	exp_round_1	exposure	Cantus	Bee brain
SA522803	E1_D3_W5_diluted	exp_round_1	exposure	Cantus	Bee brain
SA522804	E1_D3_W4_diluted	exp_round_1	exposure	Cantus	Bee brain
SA522805	E1_D3_W3_diluted	exp_round_1	exposure	Cantus	Bee brain
SA522806	E1_D3_W2_diluted	exp_round_1	exposure	Cantus	Bee brain
SA522807	E1_D3_W1_diluted	exp_round_1	exposure	Cantus	Bee brain
SA522808	E1_D3_W4	exp_round_1	exposure	Cantus	Bee brain
SA522809	E1_D3_W3	exp_round_1	exposure	Cantus	Bee brain
SA522810	E1_D3_W6	exp_round_1	exposure	Cantus	Bee brain
SA522811	E1_D3_W7	exp_round_1	exposure	Cantus	Bee brain
SA522812	E1_D3_Y7_diluted	exp_round_1	exposure	ClickPro	Bee brain
SA522813	E1_D3_Y6_diluted	exp_round_1	exposure	ClickPro	Bee brain
SA522814	E1_D3_Y5_diluted	exp_round_1	exposure	ClickPro	Bee brain
SA522815	E1_D3_Y4_diluted	exp_round_1	exposure	ClickPro	Bee brain
SA522816	E1_D3_Y3_diluted	exp_round_1	exposure	ClickPro	Bee brain
SA522817	E1_D3_Y2_diluted	exp_round_1	exposure	ClickPro	Bee brain
SA522818	E1_D3_Y1_diluted	exp_round_1	exposure	ClickPro	Bee brain
SA522819	E1_D3_Y7	exp_round_1	exposure	ClickPro	Bee brain
SA522820	E1_D3_Y6	exp_round_1	exposure	ClickPro	Bee brain
SA522821	E1_D3_Y5	exp_round_1	exposure	ClickPro	Bee brain
SA522822	E1_D3_Y4	exp_round_1	exposure	ClickPro	Bee brain
SA522823	E1_D3_Y3	exp_round_1	exposure	ClickPro	Bee brain
SA522824	E1_D3_Y2	exp_round_1	exposure	ClickPro	Bee brain
SA522825	E1_D3_Y1	exp_round_1	exposure	ClickPro	Bee brain
SA522826	E1_D3_B6	exp_round_1	exposure	Control	Bee brain
SA522827	E1_D3_B7	exp_round_1	exposure	Control	Bee brain
SA522828	E1_D3_B5	exp_round_1	exposure	Control	Bee brain
SA522829	E1_D3_B4	exp_round_1	exposure	Control	Bee brain
SA522830	E1_D3_B3	exp_round_1	exposure	Control	Bee brain
SA522831	E1_D3_B1_diluted	exp_round_1	exposure	Control	Bee brain
SA522832	E1_D3_B2_diluted	exp_round_1	exposure	Control	Bee brain
SA522833	E1_D3_B3_diluted	exp_round_1	exposure	Control	Bee brain
SA522834	E1_D3_B4_diluted	exp_round_1	exposure	Control	Bee brain
SA522835	E1_D3_B5_diluted	exp_round_1	exposure	Control	Bee brain
SA522836	E1_D3_B6_diluted	exp_round_1	exposure	Control	Bee brain
SA522837	E1_D3_B7_diluted	exp_round_1	exposure	Control	Bee brain
SA522838	E1_D3_B2	exp_round_1	exposure	Control	Bee brain
SA522839	E1_D3_B1	exp_round_1	exposure	Control	Bee brain
SA522840	E1_D3_R3_diluted	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522841	E1_D3_R1_diluted	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522842	E1_D3_R2_diluted	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522843	E1_D3_R5_diluted	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522844	E1_D3_R4_diluted	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522845	E1_D3_R6_diluted	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522846	E1_D3_R7_diluted	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522847	E1_D3_R7	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522848	E1_D3_R6	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522849	E1_D3_R5	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522850	E1_D3_R4	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522851	E1_D3_R3	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522852	E1_D3_R2	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522853	E1_D3_R1	exp_round_1	exposure	SIVANTOprime	Bee brain
SA522854	E1_D10_W6	exp_round_1	post_exposure	Cantus	Bee brain
SA522855	E1_D10_W3	exp_round_1	post_exposure	Cantus	Bee brain
SA522856	E1_D10_W4	exp_round_1	post_exposure	Cantus	Bee brain
SA522857	E1_D10_W1	exp_round_1	post_exposure	Cantus	Bee brain
SA522858	E1_D10_W7	exp_round_1	post_exposure	Cantus	Bee brain
SA522859	E1_D10_W2_diluted	exp_round_1	post_exposure	Cantus	Bee brain
SA522860	E1_D10_W1_diluted	exp_round_1	post_exposure	Cantus	Bee brain
SA522861	E1_D10_W2	exp_round_1	post_exposure	Cantus	Bee brain
SA522862	E1_D10_W5	exp_round_1	post_exposure	Cantus	Bee brain
SA522863	E1_D10_W4_diluted	exp_round_1	post_exposure	Cantus	Bee brain
SA522864	E1_D10_W3_diluted	exp_round_1	post_exposure	Cantus	Bee brain
SA522865	E1_D10_W5_diluted	exp_round_1	post_exposure	Cantus	Bee brain
SA522866	E1_D10_W6_diluted	exp_round_1	post_exposure	Cantus	Bee brain
SA522867	E1_D10_W7_diluted	exp_round_1	post_exposure	Cantus	Bee brain
SA522868	E1_D10_Y1_diluted	exp_round_1	post_exposure	ClickPro	Bee brain
SA522869	E1_D10_Y6	exp_round_1	post_exposure	ClickPro	Bee brain
SA522870	E1_D10_Y5	exp_round_1	post_exposure	ClickPro	Bee brain
SA522871	E1_D10_Y4	exp_round_1	post_exposure	ClickPro	Bee brain
SA522872	E1_D10_Y3	exp_round_1	post_exposure	ClickPro	Bee brain
SA522873	E1_D10_Y7	exp_round_1	post_exposure	ClickPro	Bee brain
SA522874	E1_D10_Y1	exp_round_1	post_exposure	ClickPro	Bee brain
SA522875	E1_D10_Y2_diluted	exp_round_1	post_exposure	ClickPro	Bee brain
SA522876	E1_D10_Y3_diluted	exp_round_1	post_exposure	ClickPro	Bee brain
SA522877	E1_D10_Y4_diluted	exp_round_1	post_exposure	ClickPro	Bee brain
SA522878	E1_D10_Y5_diluted	exp_round_1	post_exposure	ClickPro	Bee brain
SA522879	E1_D10_Y6_diluted	exp_round_1	post_exposure	ClickPro	Bee brain
SA522880	E1_D10_Y7_diluted	exp_round_1	post_exposure	ClickPro	Bee brain
SA522881	E1_D10_Y2	exp_round_1	post_exposure	ClickPro	Bee brain
SA522882	E1_D10_B4_diluted	exp_round_1	post_exposure	Control	Bee brain
SA522883	E1_D10_B3_diluted	exp_round_1	post_exposure	Control	Bee brain
SA522884	E1_D10_B1	exp_round_1	post_exposure	Control	Bee brain
SA522885	E1_D10_B5_diluted	exp_round_1	post_exposure	Control	Bee brain
SA522886	E1_D10_B6_diluted	exp_round_1	post_exposure	Control	Bee brain
SA522887	E1_D10_B7_diluted	exp_round_1	post_exposure	Control	Bee brain
SA522888	E1_D10_B2_diluted	exp_round_1	post_exposure	Control	Bee brain
SA522889	E1_D10_B3	exp_round_1	post_exposure	Control	Bee brain
SA522890	E1_D10_B4	exp_round_1	post_exposure	Control	Bee brain
SA522891	E1_D10_B2	exp_round_1	post_exposure	Control	Bee brain
SA522892	E1_D10_B5	exp_round_1	post_exposure	Control	Bee brain
SA522893	E1_D10_B6	exp_round_1	post_exposure	Control	Bee brain
SA522894	E1_D10_B7	exp_round_1	post_exposure	Control	Bee brain
SA522895	E1_D10_B1_diluted	exp_round_1	post_exposure	Control	Bee brain
SA522896	E1_D10_R5	exp_round_1	post_exposure	SIVANTOprime	Bee brain
SA522897	E1_D10_R4	exp_round_1	post_exposure	SIVANTOprime	Bee brain

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Collection:

Collection ID:	CO004581
Collection Summary:	Workers were pooled to obtain a final sample number of n = 6 per treatment per timepoint per experimental round. We pooled three brains 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. 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:	TR004597
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:

TR004597

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:	SP004594
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:

SP004594

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	AN007407	AN007408
Chromatography ID	CH005612	CH005612
MS ID	MS007099	MS007100
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:	CH005612
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:	MS007099
Analysis ID:	AN007407
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:	MS007100
Analysis ID:	AN007408
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