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Received: 22 January 2024
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Revised: 27 February 2024
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Accepted: 10 March 2024
DOI: 10.1002/arch.22104
RESEARCH ARTICLE
1
H‐NMR revealed pyruvate as a differentially
abundant metabolite in the venom glands of
Apis cerana and Apis mellifera
Xing Zheng
1
|Yanjun Liu
1
|Rongshen Wang
2
|
Mingyang Geng
3
|Jinliang Liu
4
|Zhenxing Liu
1,5
|
Yazhou Zhao
1
1
State Key Laboratory of Resource Insects,
Institute of Apicultural Research, Chinese
Academy of Agricultural Sciences, Beijing,
China
2
Shijiazhuang Animal Disease Prevention and
Control Center, Hebei, China
3
Ili Kazakh Autonomous Prefecture General
Animal Husbandry Station, Xinjiang Uighur
Autonomous Region, China
4
Beijing Shennong's Country Apiculture
Specialized Cooperative, Beijing, China
5
School of Medicine, Chongqing University,
Chongqing, China
Correspondence
Zhenxing Liu and Yazhou Zhao, State Key
Laboratory of Resource Insects, Institute of
Apicultural Research, Chinese Academy of
Agricultural Sciences, Beijing, 100093, China.
Email: liuzhenxing01@caas.cn and
zhaoyazhou0301@126.com
Funding information
China Agriculture Research System‐Bee,
Grant/Award Number: CARS‐44‐KXJ17; The
Science and Technology Innovation Project of
Chinese Academy of Agricultural Sciences,
Grant/Award Number: CAAS‐ASTIP‐
2024‐IAR
Abstract
As a common defense mechanism in Hymenoptera, bee
venom has complex components. Systematic and compre-
hensive analysis of bee venom components can aid in early
evaluation, accurate diagnosis, and protection of organ
function in humans in cases of bee stings. To determine the
differences in bee venom composition and metabolic
pathways between Apis cerana and Apis mellifera, proton
nuclear magnetic resonance (
1
H‐NMR) technology was
used to detect the metabolites in venom samples. A total of
74 metabolites were identified and structurally analyzed in
the venom of A. cerana and A. mellifera. Differences in the
composition and abundance of major components of bee
venom from A. cerana and A. mellifera were mapped to four
main metabolic pathways: valine, leucine and isoleucine
biosynthesis; glycine, serine and threonine metabolism;
alanine, aspartate and glutamate metabolism; and the
tricarboxylic acid cycle. These findings indicated that the
synthesis and metabolic activities of proteins or polypep-
tides in bee venom glands were different between A. cerana
and A. mellifera. Pyruvate was highly activated in 3 selected
metabolic pathways in A. mellifera, being much more
dominant in A. mellifera venom than in A. cerana venom.
These findings indicated that pyruvate in bee venom glands
is involved in various life activities, such as biosynthesis and
Arch Insect Biochem Physiol. 2024;115:e22104. wileyonlinelibrary.com/journal/arch © 2024 Wiley Periodicals LLC.
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https://doi.org/10.1002/arch.22104
energy metabolism, by acting as a precursor substance or
intermediate product.
KEYWORDS
1
H‐NMR, Apis cerana,Apis mellifera, bee venom, metabolomics
Highlights
•A total of 74 metabolites were detected in the venoms
of A. cerana and A. mellifera using
1
H‐NMR techniques.
•The differential compounds between the two honeybee
venoms were mainly involved in four metabolic
pathways.
•A. mellifera venom contained more pyruvate compared
to A. cerana venom.
1|INTRODUCTION
Like snake and scorpion venom, bee venom is a common defense mechanism in Hymenoptera with strong
toxicity and complex components, playing an important role in bee defense against intruders and predators
(Shi et al., 2022a). Bee venom is a fragrant, volatile, transparent liquid, which is secreted by the venom glands of
worker bees and usually stored in the venom sac. There are two venom glands associated with worker bee sting
apparatuses, namely, the acid gland, located in the abdominal cavity, and the alkaline gland, which is shorter and
thicker than the acid gland (Kheyri et al., 2013). When worker bees reach an age of approximately 14 days, the
secretion amount of bee venom begins to increase, reaching its peak at the guard and collecting stages. As worker
honeybees age, venom secretion gradually decreases until it completely disappears (Gnatzy et al., 2015). Bee venom
is acidic and contains a certain number of bioactive compounds (Maitip et al., 2021), including kinins (mast cell
degranulation peptides), bee venom peptides (Duffy et al., 2020), polypeptides, biogenic amines (catecholamines,
acetylcholine, histamine), and enzymes (cholinesterase, hyaluronidase, phospholipase, protease) (Wehbe
et al., 2019).
Metabolomics technologies can typically be categorized into targeted and untargeted approaches (Olivier
et al., 2019). Targeted strategies focus on separating and quantifying a group of predefined molecules, typically
using analytical standards. Untargeted approaches are applied to obtain a global profile of substances in a
sample and are generally used for composition identification rather than quantitative analysis (Wolfender
et al., 2019). Comprehensive qualitative analysis of substances in bee venom using high‐throughput, sensitive,
and selective omics methods can help elucidate the pharmacological significance of bee venom and optimize
treatment strategies for diseases associated with bee venom (Klupczynska et al., 2020). With the development
of new techniques such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS),
chromatography, and gene sequencing, as well as the cross‐integration of multiple omics technologies,
including proteomics, peptidomics, transcriptomics, genomics, and metabolomics, research on bee venom has
advanced (Prashanth et al., 2017). Omics technology is applied for large‐scale data collection and analysis,
especially in characterizing and quantifying overall biological molecules and their effects on biology. In
summary, omics technology has provided a new strategy for the study of bee venom components, enabling its
gradual integration into biological information big data systems (von Reumont et al., 2022). Metabolomic
technology can reveal previously unknown components in bee venom, increase the understanding of the
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ZHENG ET AL.
biological functions of bee venom components, and improve medical treatment methods for bee stings (Pawlak
et al., 2020). However, metabolomics also has some challenges, such as complexity and instability during
sample pretreatment and preparation (Klupczynska et al., 2018). Organic acids are trace components of bee
venom, and their concentrations differ widely. It has been reported that the content of quinolinic acid in bee
venom is only 0.002 mg/g, while that of citric acid can beashighas86mg/g,spanningfourordersofmagnitude
(Marković‐Housley et al., 2000). It is impossible to analyze organic acid fingerprints using the same preparation
method. Notably, the bee venom composition is a dynamic system that changes with changing internal and
external environmental conditions (De Graaf et al., 2021). Therefore, sample collection is also a key part of
metabolome research.
Due to the complex and diverse composition of bee venom, damage to target organs in humans can be
exacerbated by the synergistic effects of some components after a bee sting, which can lead to disability or
even death in severe cases (Shi et al., 2022b). Therefore, systematic and comprehensive analysis of the
components of bee venom may be crucial for early and accurate protection of organ function (Zheng
et al., 2023). Previous studies have focused mostly on the proteins and polypeptides present in bee venom, but
bee venom also contains many small molecules, such as lipids, amino acids, and sugars. Metabolomics
technology is ideal for analyzing small molecular substances (Wehbe et al., 2019). In this study, we used proton
NMR (
1
H‐NMR) spectroscopy to analyze the material composition of venom glands from 2 bee species, Apis
cerana and Apis mellifera, and identified possible differential markers and the metabolic pathways involved. This
study aimed to provide insight into the biosynthesis of active components in bee venom and the underlying
mechanism of bee sting pathogenesis.
2|MATERIALS AND METHODS
2.1 |Sample collection and preparation
Using pollen carried on the legs as a sampling marker, 150 live worker bees were randomly captured with tweezers
at the entrances of A. mellifera and A. cerana colonies, and each worker bee was placed in a 1.5 mL centrifuge tube.
There were 50 bees per repetition, for a total of three repetitions. During sampling, harm or death of the bees was
avoided. After sampling, the centrifuge tubes were immediately placed on ice to ensure that the bees were
unconscious. Using sterile pointed tweezers, the venom sacs and glands were dissected from bee tails, and the bee
species were distinguished and placed in new centrifuge tubes (Flanjak et al., 2021). The venom sacs and glands of
A. cerana (Group C) and A. mellifera (Group L) were placed in a −80°C freezer for storage before analysis, with
sufficient backup samples.
Before
1
H‐NMR analysis, the bee venom gland samples were freeze‐dried. The A. cerana and A. mellifera
bee venom gland samples were prefrozen for 3 h in a −40°C quick‐freezing chamber and then transferred to a
freeze‐drying box with a vacuum pressure of approximately 80 Pa and a cold trap temperature of
approximately −40°C. The heating plate was heated to 40°C for 30 min and then held for 840 min. After
30 min, the temperature was raised to 50°C and then held at this temperature for 120 min. After 30 min, the
temperature was lowered to 20°C, and the sample was removed and placed in a desiccator for use (Zhou
et al., 2010).
Each lyophilized bee venom gland sample was dissolved in 450 μLofD
2
O and transferred to a clean
centrifuge tube that had been prefilled with 50 μLofAnachro‐certified 2,2‐dimethyl‐2‐silapentane‐5‐
sulfonate (DSS) standard solution (ACDSS, 4.136 mM) (Frangieh et al., 2019). ACDSS was used as an internal
standard compound for
1
H‐NMR spectroscopic analysis, and its chemical shift in the
1
H‐NMR spectrum was
0.0 ppm.
ZHENG ET AL.
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2.2 |
1
H‐NMR spectroscopy analysis
Metabolites in the venom gland samples were detected using an NMR spectrometer. Each bee venom gland sample
was tested in triplicate. The NMR spectrometer was a Bruker AV III 600 MHz instrument (Bruker Biospin, Milton,
Canada), which was equipped with a reverse freezer and a working parameter of 600.13 MHz (Klupczynska
et al., 2018).
2.3 |Data analysis
The
1
H‐NMR free induction decay signal was imported into the Chenomx NMR suite (version 8.0; Chenomx,
Edmonton, Canada) software for automatic Fourier transform, phase adjustment, and baseline correction processes.
The DSS‐d6 peak (0.0 ppm) was used as the standard for all spectral chemical shifts, and this peak was subjected to
an inversion convolution operation to adjust the spectral peak shape. Based on the relevant information (such as
chemical shift, peak shape, half‐peak width, and coupling splitting) of the
1
H‐NMR spectrum, the concentration and
spectral peak area of DSS‐d6 were used as the standard, and the signals of the sample spectra were analyzed using
Chenomx's built‐in database (Ma et al., 2018). Finally, the metabolites and corresponding absolute concentration
values were obtained.
The obtained data were analyzed using receiver operating characteristic curves and box plot analysis with SPSS
20.0 software, and metabolite set enrichment analysis (MSEA) and metabolic pathway analysis were performed
using MetaboAnalyst 3.0 (http://www.metaboanalyst.ca/faces/ModuleView.xhtml).
3|RESULTS
3.1 |Metabolites in the venom glands of A. cerana and A. mellifera
The metabolic profiles of the venom gland samples of A. cerana and A. mellifera were generated using
600 MHz
1
H‐NMR spectroscopy. The overall profiles of the
1
H‐NMR spectra of the venom gland samples
from the two honeybee species were similar. The affected metabolites were matched using the Chenomx
database and NMR spectral signals. Ultimately, 74 metabolites were identified from the venom gland samples
of the two honeybee species (A. cerana and A. mellifera), including amino acids and their derivatives, organic
acids, sugars, and amines. Based on the concentration of the DSS standard and Chenomx software data,
these metabolite components were quantitatively and qualitatively analyzed. All metabolite signatures
are summarized in Table 1. Among the metabolites, 15 unique metabolites were specifically detected in the
venom glands of A. mellifera, 14 unique metabolites were exclusively detected in the venom glands
of A. cerana, and 45 shared metabolites were detected in both species (Figure 1). This finding indicated
that the types of metabolites detected via
1
H‐NMR in the venom glands of the 2 honeybee species were
similar.
We subsequently conducted substance classification analysis of 74 metabolites and found that they were
mainly esters, organic acids, amino acids, sugars, and other substances. Overall, the types of metabolites in
the venom glands of A. cerana were similar to those in the venom glands of A. mellifera,withonlyslight
differences in esters, organic acids, and sugars (Figure 2). For substance classification patterns, identifying
metabolic markers of the venom glands of A. cerana and A. mellifera is difficult because they exhibit similar
characteristics.
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TABLE 1 Metabolite signatures of honey bee venom.
No. Metabolites Formula KEGG Compound ID Apis mellifera Apis cerana
12‐Hydroxybutyrate C
4
H
8
O
3
C05984 + +
22‐Hydroxyisobutyrate C
4
H
8
O
3
C21297 + +
32‐Oxoisocaproate C
6
H
10
O
3
C00233 + ‐
43‐Aminoisobutyrate C
4
H
9
NO
2
C01205 + +
53‐Hydroxyisovalerate C
5
H
10
O
3
C20827 + ‐
63‐Methyl‐2‐oxovalerate C
6
H
10
O
3
C03465 + ‐
74‐Aminobutyrate C
4
H
9
NO
2
C00334 + ‐
8 ADP C
10
H
15
N
5
O
10
P
2
C00008 + ‐
9 Acetate C
2
H
4
O
2
C00033 + +
10 Acetone C
3
H
6
O C00207 + +
11 Adenine C
5
H
5
N
5
C00147 + +
12 Adenosine C
10
H
13
N
5
O
4
C00212 + ‐
13 Alanine C
3
H
7
NO
2
C00041 + +
14 Arginine C
6
H
14
N
4
O
2
C00062 + +
15 Benzyl benzoate C
14
H
12
O
2
C12537 ‐+
16 Betaine C
5
H
11
NO
2
C00719 + +
17 Choline C
5
H
14
NO C00114 + +
18 Creatine phosphate C
4
H
10
N
3
O
5
P C02305 + +
19 Dimethyl sulfone C
2
H
6
O
2
S C11142 + ‐
20 Dimethylamine C
2
H
7
N C00543 + +
21 Ethanol C
2
H
6
O C00469 + ‐
22 Ethanolamine C
2
H
7
NO C00189 ‐+
23 Formate CH
2
O
2
C00058 + +
24 Fructose C
6
H
12
O
6
C00095 + +
25 Fumarate C
4
H
4
O
4
C00122 + +
26 Glucose C
6
H
12
O
6
C00031 + +
27 Glutamate C
5
H
9
NO
4
C00025 ‐+
28 Glutamine C
5
H
10
N
2
O
3
C00064 + +
29 Glycine C
2
H
5
NO
2
C00037 + +
30 Guanidoacetate C
3
H
7
N
3
O
2
C00581 ‐+
31 Guanosine C
10
H
13
N
5
O
5
C00387 + +
32 Hypoxantine C
5
H
4
N
4
O+‐
33 IMP C
10
H
13
N
4
O
8
P C00130 + +
34 Imidazole C
3
H
4
N
2
C01589 + +
(Continues)
ZHENG ET AL.
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TABLE 1 (Continued)
No. Metabolites Formula KEGG Compound ID Apis mellifera Apis cerana
35 Inosine C
10
H
12
N
4
O
5
C00294 + +
36 Isobutyrate C
4
H
8
O
2
C02632 + ‐
37 Isoleucine C
6
H
13
NO
2
C00407 + +
38 Isovalerate C
5
H
10
O
2
C08262 + +
39 Lactate C
3
H
6
O
3
C00186 + +
40 Leucine C
6
H
13
NO
2
C00123 + +
41 Lysine C
6
H
14
N
2
O
2
C00047 + +
42 Maltose C
12
H
22
O
11
C00208 + ‐
43 Methanol CH
4
O C00132 + +
44 Methionine C
5
H
11
NO
2
S C00073 + +
45 Methylguanidine C
2
H
7
N
3
C02294 + ‐
46 N‐Acetylcysteine C
5
H
9
NO
3
S C06809 + ‐
47 NAD+ C
21
H
27
N
7
O
14
P
2
C00003 + ‐
48 Nicotinate C
8
H
8
N
2
O
3
C00253 ‐+
49 Oxypurinol C
5
H
4
N
4
O
2
C07599 + ‐
50 Phenylalanine C
9
H
11
NO
2
C00079 + +
51 Proline C
5
H
9
NO
2
C00148 + +
52 Propionate C
3
H
6
O
2
C00163 + +
53 Propylene glycol C
3
H
8
O
2
C00583 + +
54 Protocatechuate C
7
H
6
O
4
C00230 ‐+
55 Putrescine C
4
H
12
N
2
C00134 + +
56 Pyruvate C
3
H
4
O
3
C00022 + +
57 Sarcosine C
3
H
7
NO
2
C00213 + ‐
58 Serine C
3
H
7
NO
3
C00065 + +
59 Succinate C
4
H
6
O
4
C00042 + +
60 Taurine C
2
H
7
NO
3
S C00245 + +
61 Threonine C
4
H
9
NO
3
C00188 + +
62 Trehalose C
12
H
22
O
11
C00183 + ‐
63 Trigonelline C
7
H
7
NO
2
C01004 + +
64 Trimethylamine C
3
H
9
N C00565 + +
65 Trimethylamine N‐oxide C
3
H
9
NO C01104 + ‐
66 Tryptophan C
11
H
12
N
2
O
2
C00078 + +
67 Tyrosine C
9
H
11
NO
3
C00082 ‐+
68 Uracil C4H
4
N
2
O
2
C00106 + +
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TABLE 1 (Continued)
No. Metabolites Formula KEGG Compound ID Apis mellifera Apis cerana
69 Uridine C
9
H
12
N
2
O
6
C00299 + ‐
70 Valine C
5
H
11
NO
2
C00183 + +
71 Vanillate C
8
H
8
O
4
C06672 ‐+
72 sn‐Glycero‐3‐phosphocholine C
8
H
21
NO
6
P C00670 + ‐
73 β‐Alanine C
3
H
7
NO
2
C00041 + +
74 π‐Methylhistidine C
7
H
11
N
3
O
2
C01152 + ‐
FIGURE 1 Metabolite distribution in the venom glands of A. cerana and A. mellifera.
FIGURE 2 Classification of metabolites in the venom glands of A. cerana and A. mellifera. Based on
1
H‐NMR, 59
metabolites were detected in the venom glands of A. cerana, and 60 metabolites were detected in the venom glands
of A. mellifera. These metabolites were classified according to their types of substances and labeled with
percentages.
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3.2 |Metabolic pathway analysis
Metabolic pathway analysis of the venom gland metabolic data of A. cerana and A. mellifera was conducted using
Pathway Studio, a model organism pathway analysis tool. As shown in Figure 3, the differentially enriched metabolic
pathways of the common metabolites between the 2 species were identified. Moreover, 4 differentially enriched
metabolic pathways were identified based on the criteria of low pvalues and high pathway impact values (Table 2):
valine, leucine and isoleucine biosynthesis; the tricarboxylic acid (TCA) cycle; glycine, serine and threonine
metabolism; and alanine, aspartate and glutamate metabolism.
3.3 |Differentially abundant metabolite analysis
To identify the differentially abundant metabolites, we compared the common metabolites in the four
metabolic pathways between the two honeybee species. As shown in Figure 4, in the valine, leucine and
isoleucine biosynthesis pathway, fumarate and pyruvate were differentially abundant between the two
FIGURE 3 Analysis of metabolic pathways (the darker and larger circles, which are also near the upper right
corner, were screened as differential metabolic pathways and are marked in the figure).
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honeybee species; the content in the A. cerana group was significantly lower than that in the A. mellifera group
(p< 0.5), while the glutamate and succinate abundances were not significantly different (p>0.5). Figure 5
shows that, in the TCA cycle pathway, there was a significant difference in the fumarate and pyruvate content
between the two honeybee species; the content in the A. cerana group was significantly lower than that in the
A. mellifera group (p< 0.5), while the glucose and succinate levels were not significantly different (p>0.5). As
showninFigure6, in the glycine, serine and threonine metabolism pathway, the choline abundance significantly
differed between the two honeybee species, with significantly lower levels in A. cerana than in A. mellifera
(p< 0.5); however, betaine, phosphocreatine, glycine, guanidoacetate, creatine, and threonine levels were not
significantly different between species (p>0.5). Figure 7shows that, in the alanine, aspartate and glutamate
metabolism pathway, only pyruvate was significantly differentially abundant between the two honeybee
species; the content in the A. cerana group was significantly lower than that in the A. mellifera group (p<0.5),
while the isoleucine, leucine, and valine contents were not significantly different (p> 0.5). Among the selected
metabolic pathways, the types of metabolites were relatively similar between the two honeybee species. Only
the pyruvate content showed significant differences in the three metabolic pathways of the two honeybee
TABLE 2 Central metabolic pathways for distinguishing between A. cerana and A. mellifera.
Pathway Name p−log(p)Holm p FDR Impact
Valine, leucine and isoleucine biosynthesis 2.73E‐05 10.51 0.001091 3.82E‐04 0.66666
TCA cycle 1.11E‐04 9.1068 0.004214 9.32E‐04 0.24593
Glycine, serine and threonine metabolism 3.69E‐04 7.9045 0.013287 0.002215 0.38443
Alanine, aspartate and glutamate metabolism 9.56E‐04 6.953 0.033453 0.005018 0.40928
TCA: Tricarboxylic acid
FIGURE 4 Differential analysis of metabolites from valine, leucine and isoleucine biosynthesis (C represents A.
cerana, L represents A. mellifera; * indicates a significant difference at the p< 0.05 level).
FIGURE 5 Differential analysis of metabolites from the TCA cycle (C represents A. cerana, L represents A.
mellifera; * indicates a significant difference at the p< 0.05 level).
ZHENG ET AL.
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species. Therefore, pyruvate may serve as a biomarker for distinguishing between the venom gland metabolic
profiles of A. cerana and A. mellifera.
4|DISCUSSION
1
H‐NMR was applied to analyze the venom gland metabolomics datasets of two major honeybee species (A. cerana
and A. mellifera) in China and further explored the related differential metabolic pathways and signature
metabolites. The results also validated the feasibility of
1
H‐NMR technology for the analysis of the chemical
components of honeybee venom. Most of those previous studies focused on the peptide components in honeybee
venom, such as melittin and phospholipase A2, which are thought to be the main allergens or active components in
honeybee venom (Prashanth et al., 2017). However, little is known about the other small molecules that are active
in honeybee venom (Carpena et al., 2020). We used
1
H‐NMR technology in this study to detect components with a
molecular weight of less than 20,000 Da in honeybee venom. Our data suggested that both A. cerana and A.
mellifera contained large amounts of esters, amino acids and organic acids in their venom glands and that the types
of components were quite similar. These findings indicated that the venom of these two honeybee species
FIGURE 6 Differential analysis of metabolites involved in glycine, serine and threonine metabolism
(C represents A. cerana, L represents A. mellifera; * indicates a significant difference at the p< 0.05 level).
FIGURE 7 Differential analysis of metabolites involved in alanine, aspartate and glutamate metabolism
(C represents A. cerana, L represents A. mellifera; * indicates a significant difference at the p< 0.05 level).
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functions through similar mechanisms of damage to biological organisms (Isidorov et al., 2023). Differences may
only exist in some specific components, possibly due to the biological evolution of A. cerana and A. mellifera (Rady
et al., 2017).
Enrichment analysis of metabolic pathways involved three amino acid metabolic pathways, namely,
valine, leucine and isoleucine biosynthesis; glycine, serine and threonine metabolism; and alanine, aspartate
and glutamate metabolism. These findings indicated that the synthesis and metabolism of amino acids in the
venom glands of different honeybee species are strongly activated. The main organic components and
functionally active substances in honeybee venom are proteins or polypeptides, such as melittin and apamin
(Moreno and Giralt, 2015). In addition, branched‐chain amino acids (valine, leucine, and isoleucine) have also
been determined to be essential indicators of honeybee life activity. The absence of these proteins directly
affects the development of individuals and groups of honeybees, including their venom glands (Baracchi
et al., 2011). For example, isoleucine is an essential nutritional factor in honeybee food, accounting for more
than 4% of the protein content (Ghosh et al., 2016). These findings were consistent with the conclusion of
the present study that the key amino acid metabolic pathways were the most affected metabolic pathways in
the venom gland. The TCA cycle is a common metabolic pathway in aerobic organisms (Lande et al., 2019).
This pathway is the ultimate metabolic pathway for the three macronutrients (carbohydrates, lipids,
and amino acids) in the body and the focal point connecting carbohydrate, lipid, and amino acid metabolism
(Akram, 2014). In this study, many differentially abundant metabolites, such as amino acids, organic acids,
and sugars, were detected in the venom glands of the two honeybee species. Most of these
substances participate in the TCA cycle. Moreover, the TCA cycle plays a significant role in most organs of
the body, especially in tissues and organs, where high levels of biosynthesis and metabolism occur
(Zheng et al., 2023).
Pyruvate is a weak organic acid with two functional groups, carbonyl and carboxyl groups (Thompson, 2000).
This compound not only has carboxylic acid and ketone properties but also has the properties of an α‐keto acid,
which is the simplest α‐keto acid (carbonyl acid). Pyruvate is a three‐carbon keto acid produced in vivo and is the
final product of the glycolysis pathway (Zhu et al., 2019). This compound can be reduced to lactic acid for energy in
the cytoplasm or oxidized to acetyl‐CoA in mitochondria, where it enters the TCA cycle and is oxidized to form
carbon dioxide and water, completing the aerobic oxidation of glucose for energy supply (Chen et al., 2021).
Pyruvate can also achieve the mutual transformation of sugar, fat and amino acids through acetyl‐CoA and the
tricarboxylic acid cycle (Hu et al., 2016). Therefore, pyruvate plays an important role as a hub in the metabolic
connection of the three major nutrients (Vassella et al., 2004). In the present study, pyruvate was found to be
activated in three differential metabolic pathways and was found to be a differentially abundant venom gland
metabolite between A. cerana and A. mellifera. These findings indicated that pyruvate participates in various life
activities in venom glands and is an important precursor substance or intermediate product for material synthesis
and energy metabolism.
5|CONCLUSION
In summary, this study found that the venom metabolites of A. cerana and A. mellifera are different. Four metabolic
pathways were mainly affected: valine, leucine and isoleucine biosynthesis; glycine, serine and threonine
metabolism; alanine, aspartate and glutamate metabolism; and the tricarboxylic acid cycle. Especially, pyruvate was
found to be higher in the venom of A. mellifera. Pyruvate can be used as a differentiator between the venom of A.
cerana and A. mellifera. These results may provide insights for the analysis of complex components of bee venom
and the mechanism of bee sting injuries.
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AUTHOR CONTRIBUTIONS
Xing Zheng and Yanjun Liu contributed equally to this work. Xing Zheng: Conceptualization, Methodology,
Software, Writing‐Original draft preparation. Yanjun Liu: Writing‐Original draft preparation, Investigation,
Validation. Rongshen Wang: Data curation and Validation. Mingyang Geng: Formal analysis and Supervision.
Jinliang Liu: Writing review and editing. Zhenxing Liu: Visualization, Supervision, Writing review and editing. Yazhou
Zhao: Project administration, Resources, Funding acquisition. All authors have read and agreed to the published
version of the manuscript.
ACKNOWLEDGMENTS
This study was supported by the earmarked fund for China Agriculture Research System‐Bee (CARS‐44‐KXJ17),
and the Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS‐ASTIP‐
2024‐IAR).
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article. Further inquiries can be directed to the
corresponding author.
ORCID
Xing Zheng http://orcid.org/0000-0003-0896-2733
Yanjun Liu http://orcid.org/0000-0001-5968-3932
Zhenxing Liu http://orcid.org/0000-0001-8236-1931
Yazhou Zhao http://orcid.org/0009-0007-5169-518X
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How to cite this article: Zheng, X., Liu, Y., Wang, R., Geng, M., Liu, J., Liu, Z. et al. (2024) 1H‐NMR revealed
pyruvate as a differentially abundant metabolite in the venom glands of Apis cerana and Apis mellifera.
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