Proteome-metabolome profiling of wax gland complex reveals functional changes in honeybee, Apis mellifera L

Rumeng Xu; Beibei Ma; Yiying Yang; Xuancheng Dong; Jianke Li; Xiang Xu; Yu Fang

doi:10.1016/j.isci.2024.109279

. 2024 Feb 20;27(3):109279. doi: 10.1016/j.isci.2024.109279

Proteome-metabolome profiling of wax gland complex reveals functional changes in honeybee, Apis mellifera L.

Rumeng Xu ^1,², Beibei Ma ^1,², Yiying Yang ¹, Xuancheng Dong ¹, Jianke Li ¹, Xiang Xu ^1,^∗, Yu Fang ^1,^3,^∗∗

PMCID: PMC10933454 PMID: 38482490

Summary

Wax gland complex (WGC) serves as the primary generator of beeswax; however, the dynamic biological function in wax secretion remains unclear. To elucidate the developmental mechanism of WGC, we conducted a comprehensive analysis to reveal the variations in proteins and metabolites among the newly emerged bee (NEB), wax-secreting bee (WSB), and overaged bee (OAB). We identified 3,295 proteins and 159 metabolites in WGC. Specifically, NEB elevated the expression of ribosomal proteins for preparing the glandular organ. While WSB promoted the size of epidermal cells and oenocytes, the enrichment of fatty acids and energy metabolism in WSB suggested a strong ability in wax synthesis. In OAB, disorganized wax tubules, and up-regulated cysteine proteases reflected the gland degeneration. These findings highlight the dynamic changes in the level of molecule and morphological structure in WGC, offering valuable insights into the development and mechanism of wax secretion in honeybees and other wax insects.

Subject areas: Entomology, Evolutionary biology, Metabolomics, Proteomics, Zoology

Graphical abstract

Highlights

•
Wax secretion ability fits the dynamic changes in the wax gland complex
•
The wax-secreting bee exhibits the strongest activity in energy and fatty acid
•
The wax gland in overaged bee undergoes organ aging and wax secretion degeneration

Entomology; Evolutionary biology; Metabolomics; Proteomics; Zoology

Introduction

As a nest-dwelling insect, honeybee (Apis mellifera L.) produce wax to construct double-sided hexagonal nest chambers.¹^,² Beeswax-built hives serve multiple functions, such as pollen storing, honey sealing, and larvae housing. In addition, comb wax plays a vital role in kin recognition within honeybee colonies and enables chemical communication among bees.³ Beeswax ranges in color from yellow to dark brown and has a pleasant aroma reminiscent of honey and pollen. It is composed of hydrocarbons, fatty acids, wax esters, and fatty alcohols.⁴ The hydrocarbons and fatty acids comprise a substantial component of beeswax, and their composition varies based on the age of the worker bees.⁵ Fatty acids play a crucial role in insect growth, development, and information exchange. Their biosynthesis involves fatty acid synthase, fatty acyl-CoA reductase, and elongase to produce long-chain fatty acid.⁶

The wax gland complex (WGC), located bilaterally in the abdomen, is a typical secretory gland responsible for beeswax synthesis in honeybees.⁷ The synthesis of beeswax is an energy-intensive process, requiring approximately 20 g of honey to produce 1 g of beeswax.⁸ Similar to other secretory glands of honeybees, such as the hypopharyngeal glands⁹ and salivary glands,¹⁰ the development of the WGC experiences dynamic changes according to age.¹¹ The peak period of WGC secreting beeswax is 10–18 days after a new bee emerges.¹² However, once worker bees reach the age of 21 days, the glands begin to deteriorate with the secretion activity decreasing.¹³ Not only in bees but also in other wax-secreting insects, wax production is closely correlated to the development of secretory glands. For instance, the secretion activity of the wax gland in Matsucoccus matsumurae and Friesella schrottkyi is significantly varied at different developmental stages.¹⁴^,¹⁵ Morphologically, the structure of WGC changes in different developmental stages of honeybees.¹¹^,¹³ In young bees, the epidermal cells are cuboidal and closely packed.¹⁶ The epidermal cells become slender and partially divided by intercellular spaces while WGC developing. Finally, the cell membranes, nuclei, and protoplasm become less defined as well as cell height decreases in the WGC of older bees.¹⁷ Therefore, a dynamic change in the wax gland’s structure correlates with the different stages of bee development and wax production.

Although the anatomical structure of the honeybee WGC has been extensively studied, the molecular mechanics during the dynamic process of WGC remain elusive. To achieve this, we employed a state-of-the-art combined approach of histological structure, and proteomic/metabolomic profile on newly emerged bee (NEB), wax-secreting bee (WSB), and the overaged bee (OAB). The comprehensive approach has been previously applied to investigate honeybee caste differentiation,¹⁸^,¹⁹ honeybee tissues or organs,²⁰ and the division of labor in worker bees.²¹ Therefore, we used this strategy to investigate the physiological changes in the WGC of honeybees. This not only advances scientific knowledge of the beeswax formation process but also has the potential for commercial production and application of beeswax.

Results and discussion

To gain insights into the molecular mechanisms underlying the development of the WGC in A. mellifera, we utilized label-free quantitative proteomics and untargeted metabolomics technologies to explore the dynamic changes in NEB, WSB, and OAB. Firstly, we identified a total of 3,295 proteins and 159 metabolically characterized ions in WGC (See Tables S1 and S2). Furthermore, we selected key proteins and metabolites involved in regulating the development of WGC, which provided an in-depth understanding of the biochemical processes driving wax formation. Morphologically, the most developed wax tubules and epidermal cells in WGC improved the secretory ability in WSB. These stage-specific patterns of proteins and metabolites provide an intensive insight into the functional activity of WGC at each developmental stage.

Core proteome and metabolome providing the material-energetic basis for WGC development

Glandular development involves cell differentiation and proliferation. To gain a systematic understanding of the fundamental regulators and biological pathways that govern the development of WGC, we analyzed the proteins and metabolites shared across the three different aged WGC. The core proteome, including 1,027 proteins during the development of the WGC, represented 31.2% of the proteins identified (Figure 1A; Table S3). The core proteome primarily consists of proteins related to protein translation, energy metabolism, and folding/degradation (Figures 1B and 1C; Table S4). There were 65 metabolites co-expressed by the NEB-WSB and NEB-OAB (Figure 1D; Table S5), which were significantly enriched (p < 0.05) in arginine and proline metabolism, aminoacyl-tRNA biosynthesis, and glycine, serine, and threonine metabolism pathways (Figure 1E; Table S5).

Proteins and metabolites expressed in wax gland complex of NEB, WSB, and OAB

(A) Comparison of the proteins identified in WGC at NEB, WSB, and OAB.

(B) ClueGO analysis showed the core proteins identified in the WGC of NEB, WSB, and OAB. The significantly enriched functional gene ontology categories in biological processes were determined by comparing the input data with the background of gene ontology annotations in the honeybee genome using a right-sided hypergeometric test. The nodes in functionally grouped networks were connected based on a kappa score of 0.4. The FDR was controlled using a Bonferroni step-down test to adjust the p value of GO terms. The use of single and double asterisks signifies significant enrichment at the 0.05 and 0.01 levels of statistical significance, respectively.

(C) KEGG analysis was performed to identify the core proteins present in the WGC of NEB-WSB-OAB.

(D) Overlap of the putative metabolites identified from the WGC of NEB, WSB, and OAB.

(E) Ninety-nine differential metabolites were screened based on VIP >1, p value <0.05, and |log₂FC| > 1 principles (Table S5). Enrichment and analysis were carried out on 65 of the 77 differential metabolites expressed in both NEB-WSB (Table S5), as well as 87 differential metabolites observed in NEB-OAB (Table S5).

The core proteome is responsible for supplying essential energy for cell growth and tissue development in the WGC. The core proteins involved in glycolysis, pyruvate metabolism, fatty acid degradation, ATP metabolism, ribosome and protein folding (Figures 1B and 1C; Table S4), which contribute to energy metabolism and protein synthesis. Generally, glycolysis breaks down glucose to produce ATP and pyruvate,²² which is metabolized to cellular energy ATP further.²³ Fatty acid catabolism also provides ATP, especially during periods of low glucose availability.²⁴ Therefore, these processes provide a sustainable energy supply to drive WGC development. Furthermore, the translational elongation pathway is a process for new protein synthesis, whereas the pathway of protein folding refers to the structural fitting and conformation.²⁵ For instance, elongation factor 1-gamma and elongation factor 1-beta are related to the polypeptide elongation process of protein synthesis,²⁶ and elongation factor eEF1A plays a role in regulating cytoskeletal organization.²⁷ Beyond their roles in energy metabolism and protein synthesis, core proteins engage in diverse metabolic processes. They also protect cells from harmful substances, particularly in antioxidant activity.²⁸^,²⁹ Peroxiredoxin, a cytoprotective antioxidant enzyme, acts as a defense against endogenous or exogenous peroxide attacks.³⁰ These functions indicate the diversity and complexity of core proteins in cellular physiology, emphasizing their central role in maintaining cell and organismal health during the development of WGC.

Similar to the core proteome, the essential metabolome also showed a significant metabolic pathway involved in energy, translation, and protein biosynthesis (See Table S5). For instance, amino acids play a vital role in transferring to ribosomal synthetic proteins through aminoacyl-tRNA biosynthesis.³¹^,³² Arginine, in particular, contributes to various biological functions, including protein synthesis, urea production, and the metabolism of glutamate and proline.³³ In conclusion, the core proteome and metabolome play crucial roles in energy support, tissue construction, beeswax synthesis, and antioxidant defense during WGC development.

WGC development preparation in NEB

Proteins expressed specifically during a distinct stage provide key insights into the unique biological processes and pathways. Totally, 1,910 proteins were identified in NEB (See Table S3), which were associated with peptide metabolic process, protein localization, actin filament organization, ribonucleoside monophosphate metabolic, mRNA surveillance pathway, and protein export (Figures 4A and 4B; Table S6). The mRNA surveillance pathway plays a crucial role in identifying and degrading abnormal mRNAs, preventing the production of harmful proteins.³⁴ Actin provides structural support to cells and is a critical component of the cytoskeleton. The dynamic polymerization and depolymerization of actin filaments determine cell shape and structure, which are integral to various cellular functions.³⁵ These processes contribute to the development of the WGC, as the mRNA signaling regulatory pathway plays a role in maintaining cell structure, normal production of functional proteins in WGC, and promoting cell motility. Actin filaments provide the necessary cellular infrastructure for the protein synthesis machinery and are involved in transporting these proteins within the cell.

Enrichment analysis of specifically expressed proteins in NEB, WSB, and OAB

(A) NEB-specific expressed protein gene ontology (GO) analysis.

(B) NEB-specific expressed protein Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.

(C) WSB-specific expressed protein GO analysis.

(D) WSB-specific expressed protein KEGG analysis.

(E) OAB-specific expressed protein GO analysis.

(F) OAB-specific expressed protein KEGG analysis.

The dynamic changes of secretory cells are synchronized with the organization of secretory glands. The cells in WGC, including epidermal cells, oenocytes, and adipocytes, work together to secrete a wax mixture of hydrocarbons, fatty acids, and lipoproteins.¹¹ Unexpectedly, the structure of WGC had been organized to some extent in the initial phase of the honeybee. There were a large number of cells in WGC already (Figure 3C), even though the cells were not at their maximum. The surface of wax mirror appeared smooth with no visible lipid droplets (Figures 2A and 3A). Furthermore, the wax tubules, essential for wax transportation, were orderly yet sparsely distributed, stretching out toward the wax mirror’s surface (Figure 3B). These morphological features align with previous findings.¹¹^,³⁶ Inherently, the structural changes within the cell result in functional shifts that are key to the intricate developmental processes observed in the WGC. The initial organized pattern of the WGC organized in NEB is supported by the 414 up-regulated proteins involved in energy metabolism, structural integrity, and protein synthesis (Figures 5A and 5B; Tables S7 and S8). For example, F-actin capping proteins and ribosomal proteins maintain cellular structure and support protein synthesis.²⁵^,³⁷

The inner structure of WGC in NEB, WSB, and OAB

Panel A, D, and G are captured with scanning electron microscope, panel B, C, E, F, H, and I are with transmission electron microscope.

(A) Wax mirror surfaces during the period of NEB. Scale bar, 20 μm.

(B) Epidermis during the period of NEB. Scale bar, 1 μm.

(C) Change in other cells within the gland during the period of NEB. Scale bar, 10 μm.

(D) Wax mirror surfaces during the period of WSB. Scale bar, 20 μm.

(E) Epidermis during the period of WSB. Scale bar, 1 μm.

(F) Change in other cells within the gland during the period of WSB. Scale bar, 10 μm.

(G) Wax mirror surfaces during the period of OAB. Scale bar, 20 μm.

(H) Epidermis during the period of OAB. Scale bar, 1 μm.

(I) Change in other cells within the gland during the period of OAB. Scale bar, 10 μm.

The surface of the wax mirror in NEB, WSB, and OAB

(A) Smooth the surface of the wax mirror in NEB. Scale bar, 500 μm.

(B) Holes on the surface of the wax mirror that transport lipid droplets in three periods. Scale bar, 5 μm. Panel C, D, E, and F occurs during the WSB period.

(C) Wax lipid droplets accumulate on the surface of the wax mirror. Scale bar, 20 μm.

(D) Wax lipid droplets continue to accumulate. Scale bar, 10 μm.

(E) Formation of wax scales. Scale bar, 500 μm.

(F) Wax scales on the surface of wax mirror. Scale bar, 1 mm.

Functional analysis of up-regulated proteins expressed in NEB, WSB, and OAB

(A) GO analysis of up-regulated proteins in NEB.

(B) KEGG analysis of up-regulated proteins in NEB.

(C) GO analysis of up-regulated proteins in WSB.

(D) KEGG analysis of up-regulated proteins in NEB.

(E) GO analysis of up-regulated proteins in OAB.

(F) KEGG analysis of up-regulated proteins in OAB.

Metabolites up-regulated in NEB were involved in biological pathways such as phenylalanine, tyrosine, and tryptophan biosynthesis, ubiquinone and terpenoid quinone synthesis, and phenylalanine metabolism (Figure 7A; Tables S9 and S11). As a sole precursor of tyrosine,³⁸ phenylalanine is necessary for growth and metabolic functions as well.³⁹ Ubiquinone and terpenoid quinones participate in electron transfer and oxidative phosphorylation processes within cells, crucial for energy production.⁴⁰^,⁴¹ These findings indicate elevated metabolic activity in terms of protein synthesis and high-energy metabolism in the gland, which correspond to the proteomic results. The significant up-regulation (p < 0.05) of both protein and metabolite expression involved in energy metabolism, along with the increased number of cells within the WGC, suggest that metabolic activity during this stage primarily aims to provide sufficient energy and raw materials to support gland cell growth and division.

Enrichment of the up-regulated and expressed metabolites in NEB-WSB

The data were analyzed using the oebiotech online cloud platform (https://www.oebiotech.com/index.php?c=show&id=414). Metabolites meeting the criteria of VIP>1 were analyzed via multivariate modeling of VIP values.

(A) Enrichment was analyzed for differential up-regulated expression of 21 metabolites by NEB in NEB-WSB.

(B) Enrichment was analyzed for differential up-regulated expression of 56 metabolites by WSB in NEB-WSB.

Lipid conversion in WGC of WSB

The process of wax secretion is mainly to produce lipids. Compared to NEB, the wax lipid droplets were found to be widely dispersed on the wax mirror surface in WSB (Figures 2C and 2D). These droplets are synthesized by oenocytes and adipocytes and then transported to the wax mirror surface through pores (Figure 2B).¹¹^,¹³ Subsequently, oval wax scales, corresponding to the profile of the wax mirror, were formed by sequential embedding and solidification of thin layers on the wax mirror surface (Figures 2E and 2F). The thickness of the wax scales directly correlates with the WGC developed in the abdomen of bees. Generally, the scale could be used in constructing comb cells by bees when the thickness reaches 200–500 μm.¹⁷ The wax tubules, which are responsible for transporting lipid droplets, were well-aligned and distributed with greater density compared to the morphological characteristics in NEB and OAB (Figure 3E). Furthermore, the nuclei of oenocytes and adipocytes were distinguishable, with a significant increase in cell volume (Figure 3F). The cellular and tissue profiles match the fact that well-structured plasma membrane reticular system (PMRS) and glycogen storage rise during wax synthesizing.⁴² A well-developed PMRS promotes the activities of protein and lipid synthesis in the plasma membrane of WGC.⁴³ Therefore, the cellular structure in WGC facilitated the efficient secretion of wax in WSB.

WSB exhibited a robust metabolic ability during the peak time of their wax gland development. A total of 865 proteins were identified in WSB (See Table S3), which were enriched in energy metabolic processes, including the organonitrogen compound biosynthetic process, fatty acid biosynthesis, and oxidative phosphorylation (Figures 4C and 4D; Table S6). The process of beeswax secreting requires a significant amount of energy, as approximately 20 g of honey are equivalent to 1 g of wax.¹³^,⁴⁴ To achieve the conversion, pathways related to energy metabolism were launched. Oxidative phosphorylation and fatty acid biosynthesis dominate the production of ATP,⁴⁵ which is essential for the process of wax secretion.⁴⁶ Beyond that, the latter process could generate maleyl coenzyme A, which is an essential precursor for the synthesis of long-chain fatty acids⁴⁷ in the material stocking of beeswax.

The WGC presents an efficient conversion of lipid from glucose in WSB, which is reflected in the overrepresented proteins with elevated expression in WSB compared to the other phases. Quantitative analysis showed that 83 proteins were selected as significantly (Fold change ≥1.5, false discovery rate (FDR) ≤1, p < 0.05) up-regulators in WSB (See Table S7), which were related to mitochondrial ATP synthesis coupled with electron transport, tricarboxylic acid cycle, energy derivation by oxidation of organic compounds, generation of precursor metabolites and energy, and oxidative phosphorylation (Figures 5C and 5D; Table S8). Specifically, TCA, as a core oxidative pathway for intermediary metabolism,⁴⁸ and the oxidative phosphorylation process, involving proteins such as cytochrome c oxidase subunit, acyl carrier protein, and ATP synthase subunit d, play crucial roles in ATP production in mitochondria.⁴⁹ Notably, the activated energy metabolism in wax secretion is also identified in other insects.¹⁴^,⁵⁰ The stored energy in honeybees, such as fat and glycogen, is mobilized and converted to support wax synthesis during the process of wax secretion,⁵¹ during which the intracellular oxidative stress is generated. Therefore, the up-regulated protein (compared with OAB) superoxide dismutase 1, which was validated by western blotting (Figure 6B), and the elevated expressed metabolite L-glutathione (reduced) act as antioxidant effectors to maintain intracellular ion homeostasis.⁵⁰^,⁵²^,⁵³ Biological processes and energy expenditure have a major impact on beeswax biosynthesis, while important enzymes of fatty acid synthesis are central. Fatty acyl-CoA reductase catalyzes the conversion of fatty acids into fatty alcohols.⁵⁴ The significantly up-regulated expression (Fold change ≥1.5, FDR ≤1, p < 0.05) of fatty acyl-CoA reductase and long-chain specific acyl-CoA dehydrogenase in WSB (See Table S7) reflected the dependence of bees on specific metabolic pathways, particularly those involved in fatty acid metabolism and fatty alcohol production, for beeswax secretion. Similarly, fatty acid synthase and fatty acid elongase are key enzymes for fatty acid synthesis, and their changes at different developmental stages of Matsucoccus matsumurae were found to be closely related to the wax secretion activities of the insects.⁵⁵ The metabolites related to aminoacyl-tRNA biosynthesis pathway were significantly expressed (p < 0.05) in WSB in comparison with NEB (Figure 7B; Table S11). Aminoacyl-tRNA biosynthesis facilitates the transfer of amino acids for ribosomal protein synthesis.³¹^,³² Thus, WSB has a greater ability to synthesize proteins compared to non-wax-secreting bees. Additionally, as the key regulator in the processes of lipid transport and metabolism,⁵⁶ NPC intracellular cholesterol transporter 2 was expressed significantly (Fold change ≥1.5, FDR ≤1, p < 0.05) in WSB (compared with OAB), which was validated by western blotting (Figure 6C). Collectively, these results highlight dynamic expressions of proteins and metabolites within WSB reflecting essential adaptive changes during the booming wax production.

Western blotting analysis of superoxide dismutase1 (SOD1), NPC intracellular cholesterol transporter 2 (NPC2), and cysteine proteinase EP-B2 (EPB2)

The protein samples from the WGC of NEB, WSB, and OAB (*Apis mellifera*) are subjected to SDS-PAGE followed by western blotting analysis. SOD1, NPC2, and EPB2 are detected using the corresponding polyclonal antibodies. β-actin is used as a reference control.

(A) The western blotting bands of SOD1, NPC2, EPB2, and β-actin.

(B−D) The relative expression values of SOD1, NPC2, and EPB2 in the WGC at the three stages (normalized by β-actin). The error bar is the standard deviation.

Senescence of WGC in OAB

The structure of the WGC takes place with dramatic changes in OAB. To be specific, a few fragments of wax scales are distributed on the surface of the wax mirror, and the arrangement of wax tubules became looser and thinner (Figures 3G and 3H). Furthermore, the presence of lysosomes and intracellular vacuolization, together with the reduction in oenocyte and adipocyte number indicated the glands were degenerating (Figure 3I), which is consistent with previous work.¹⁶

The degeneration of the gland is accompanied by changes in protein expression. During the degenerative stage of the WGC, proteins expressed in OAB were enriched in actin cytoskeleton organization, aerobic respiration pathway, pyruvate metabolism, and glycerophospholipid metabolism (Figures 4E and 4F; Table S6). Particularly, the integrity of the cytoskeleton, dysregulation, or disruption of actin skeleton reorganization could lead to cell signaling malfunctions and cell death, which might accelerate glandular aging.⁵⁷ Moreover, the efficiency of aerobic respiration may result in energy yield decreasing and oxidative stress increasing.⁵⁸ Furthermore, the 17 proteins with expression elevated significantly (Fold change ≥1.5, FDR ≤1, p < 0.05) in OAB (See Table S7) were enriched in iron ion transmembrane transport pathway (Figures 5E and 5F; Table S8). Iron ions play a role in the generation of reactive oxygen species (ROS),⁴⁹ excess unstable iron ions catalyze the production of reactive oxygen species ROS and lipid peroxidation, which drive toxic biological processes in cells of WGC.⁵⁹ The increased expression of cysteine proteases may promote apoptosis in wax gland cells,⁶⁰ which was verified by western blotting (Figure 6). As a significant protein in lysosomes, cysteine protease plays a crucial role in the regulation and execution of apoptosis.⁶¹ These findings reveal the complexity of protein changes during WGC senescence and provide important biological insights for a further comprehensive understanding of wax gland development.

Metabolically, a total of 87 differential metabolites (VIP score >1, p value <0.05, fold change >2) were detected in the NEB-OAB, which included organic compounds, nucleotides, flavonoids, and vitamins (See Table S10). Seven metabolic pathways were significantly enriched (p value <0.05), including glycine, serine, and threonine metabolism, purine metabolism, and arginine and proline metabolism (See Figure S2B). As glands aging, an excess of amino acids could be transformed into essential molecules for energy production, such as pyruvate acid and acetyl coenzyme A.⁶²^,⁶³ Moreover, the metabolic changes in neurotransmitters, such as acetylcholine (See Table S10), may affect the nervous system’s capacity to control the glands, making the individual more vulnerable to hormonal influences.⁶⁴ Consequently, these modifications may hinder the lifespan of the glands, contributing to the aging process.

Conclusion

We present a systematic and comprehensive compilation of information on the morphological, structural, and functional changes in the honeybee WGC throughout its development. The WGC cells increase their numbers in NEB, and their internal proteins and metabolites promote the functional development of the organs. For WSB, the organ structures are well developed, and the energy metabolism and fatty acid synthesis proteins in the glands are active, resulting in the highest wax secretion ability. In OAB, glandular vacuolation, scattered wax tubules, and increased expression of cysteine proteases were observed, indicating aging of the wax gland complex and a decrease in wax secretion. These findings reveal the dynamic changes in wax gland function during the wax secretion of honeybees. This work advances the understanding of WGC dynamic changes and the wax secretory process, as well as and ecological roles of insects, which lays a theoretical foundation for the development and utilization of new energy and sustainable resources in the future.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Anti-Superoxide Dismutase 1 antibody [EP1727Y]	Abcam	Cat#AB51254
Cysteine Proteinase EP-B2 (EPB2) Antibody (HRP)	Abbexa	Cat#abx319501
NPC2 Polyclonal Antibody	Thermo Fisher Scientific	Cat# PA5-28955; RRID:AB_2546431

Chemicals, peptides, and recombinant proteins

C18 solid-phase extraction cartridges	CDS Analytical LLC	Cat#4115SD
Sequencing grade trypsin	Promega	Cat#V5113
Protease inhibitor (cOmplete, EDTA-free)	Roche	Cat#05 892 791 001

Deposited data

Raw data	ProteomeXchange Consortium	http://proteomecentral.proteomexchange.org

Software and algorithms

ImageJ
Peaks Studio 8.5	Ma et al.⁶⁵	https://imagej.net/software/imagej/
Cytoscape	Bindea et al.⁶⁶	https://cytoscape.org/
ClustVis	Metsalu et al.⁶⁷	https://biit.cs.ut.ee/clustvis/
SIMCA 14.1	This paper	https://www.sartorius.com/en/products/process-analytical-technology/data-analytics-software/mvda-software/simca
Oebiotech online cloud platform	This paper	https://www.oebiotech.com/index.php?c=show&id=414

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Resource availability

Lead contact

Further information and requests for resources should be directed to the lead contact, Yu Fang, fangyu@caas.cn.

Materials availability

This study did not generate new unique reagents.

Data and code availability

•
All data reported in this paper will be shared by the lead contact upon request.
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

Experimental model and study participant details

The honey bee (Apis mellifera L.) colonies used for sampling were raised in the apiary (Beijing) of the Institute of Apicultural Research, Chinese Academy of Agricultural Sciences. The samples were harvested from May to July when there were abundant sources of nectar and pollen available. NEBs were selected from 5 colonies with similar strength and painted on their thoraxes. Subsequently, NEB (1 day old), WSB (bees working on wax production and hive construction), and OAB (bees living in hives after foraging activities) were collected (600 bees in each colony) and stored at -80°C until further experiments.

Method details

WGC dissection

According to the protocol described previously,⁶⁸ WGC samples of worker bees were dissected using a stereoscope (Leica, Wetzlar, German). The abdominal plates were separated from the sting and intestinal tract with forceps, then extracted the ventral segments 3-6 using scissors.¹¹ Finally, the WGC samples were stored at -80°C.

WGC ultrastructure

To understand the tissue structure at the cellular level of WGC at different developmental stages, we employed a scanning electron microscope (SEM) and transmission electron microscope (TEM) for morphological analysis. For the preparation of SEM, WGC samples were fixed in 4% glutaraldehyde for 48 hours. After three rinses in 0.1 mol/L phosphate buffer, the complexes were dehydrated in a series of ethanol immersions: 70%, 80%, 90%, and 100% v/v, each for 5 minutes, followed by three additional immersions in 100% ethanol. The ethanol was replaced with liquid carbon dioxide before drying the samples in the EMS 850 critical point dryer. Subsequently, the samples were mounted on copper stubs, gold plated in the sputter coater, and scanned from various angles at 25 kV using a JSM-35C SEM (JEOL, Japan).

For TEM analysis, WGC samples were rinsed twice with 0.2 M PBS after removal from 2.5% glutaraldehyde and then fixed in 1% osmium tetroxide for 1 to 2 h. After another round of rinsing with 0.2 M PBS, the samples were dehydrated using a series of acetone concentrations, ranging from 10% to 100% (v/v). Next, the samples were impregnated with an embedding medium with an acetone/embedding medium ratio of 1:2 for 3 h, 2:1 for 1.5 h, and pure embedding medium for 2 h. Finally, the WGC samples were embedded in Epon 812, and the sections of semi-thin (1 mm) and ultra-thin (0.07 mm). The sections were double stained with 3% uranyl acetate and lead citrate and examined using the H-7500 TEM (HITACHI, Japan) at 80 kV.

Protein extraction

Proteins were extracted following the description previously.⁶⁵ Briefly, 50 mg WGC samples with three biological replicates were initially mixed with 500 μL lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-base, 30 mM DTT) for homogenation. Subsequently, the homogenates were centrifuged at 4°C, 12,000 g for 20 min (Microfuge 22R Centrifuge, Beckman CoulTER, US). The supernatant was added to 1 mL of cold acetone. The protein precipitate was obtained after centrifugation at 12,000 g for 20 min at 4°C. Eventually, protein pellets were resuspended by 50 μL of 5 M urea and were measured by the Bradford assay to determine the concentration.⁶⁹

Proteolytic digestion

To reduce the number of denatured proteins and prevent reformation of disulfide bonds, the samples were exposed to DTT (final concentration 10 mM) and iodoacetamide (final concentration 50 mM) successively. The sample was digested by trypsin (Promega, Madison, WI) at 37°C for 14 h according to the ratio 1:65 (enzyme: protein). Then the reaction was stopped by the addition of 1 μL of FA. The digested peptides were desalted using C18 solid-phase extraction cartridges. The eluted peptide solution was then dried using a vacuum centrifugal concentrator (Jiaimu, Beijing, China) and reconfigured in 50 μL of 0.1% FA. Peptide concentrations were analyzed using the NanoDrop 2000 spectrophotometer, and samples were diluted to 0.25 μg/μL with 0.1% FA for subsequent analysis.

LC-MS/MS analysis of proteins

HPLC-MS/MS analysis was performed on an EASY-nLC 1200 system coupled with a Q-Exactive MS (Thermo Fisher Scientific, USA). The final sample loading amount was 2 μg. Peptide samples of 8 μL were loaded onto an Acclaim PepMap 100 C18 trap column, followed by separation on an analytical column with specific specifications. The separation was carried out at a flow rate of 350 nL/min, and a 120-minute gradient elution was employed using a mobile phase comprising 0.1% FA (Phase A) and 80% acetonitrile with 0.1% FA (Phase B). The elution gradient consisted of different percentages of mobile phase B during specific time intervals (0-5min, 3%-8% B; 5-85 min 8%-20% B; 85-105min, 20%-30% B; 105-110 min, 30%-90% B; and 110-120 min 90%-90% B). The mass spectrometer utilized a full MS/ddMS2 mode, with peptide spectra acquired in the m/z range of 350-1500 and a resolution of 120,000 for the full MS scan. Moreover, ddMS2 spectra were acquired with specific parameters, including a resolution of 15,000, an isolation window of 2.0 m/z, and a normalized HCD collision energy of 28%.

Validation

To confirm the expression of results in protein levels, western blotting was employed for the validation of superoxide dismutase1 (SOD1, Abcam, USA), NPC intracellular cholesterol transporter 2 (NPC2, Invitrogen, USA), and cysteine proteinase EP-B2 (EPB2, Abbexa, UK). The protein samples, at a concentration of 2 μg/μL, were denatured in 6 × protein loading buffer and subjected to heat at 100°C for 10 min. They were then separated on 12%, and 15% SDS-PAGE gels and transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot apparatus (Thermo Fisher, USA). Subsequently, the membrane was blocked in a solution of Tris-buffered saline containing 0.5% Tween-20 and 0.5% skimmed milk for 1 h. Anti-superoxide dismutase 1 antibody (dilution ratio of 1:10000), anti-cysteine proteinase EP-B2 (EPB2) antibody (dilution ratio of 1:2500), and NPC2 polyclonal antibody (dilution ratio of 1:800) as primary antibodies were used, with an overnight incubation at 4°C, followed by treatment with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (dilution ratio of 1:5000). The immunoreactive protein bands were detected using the ECL western blotting substrate (Pierce, IL, USA) and quantified using Image J software (National Institutes of Health, USA). The protein abundance was normalized by β-actin (Sigma-Aldrich, MO, USA). The student t-test was used for statistical analysis of protein abundance.

Extraction of WGC metabolites

Untargeted metabolomics analysis was performed to investigate changes in small-molecule compounds in the WGC. Six replicates of 20 bees each of NEB, WSB, and OAB were taken per treatment. The specimens were placed on ice for manipulation. Then, 200 μL of 80% methanol was applied to each biological replicate for thorough grinding. In addition, 300 μL of 80% methanol was added to rinse the grinding head. The sample was ultrasound treated for 20 min and centrifuged at 12,000 g for 20 min at 4°C. The supernatant was filtered through a 0.22 μm membrane for the following HPLC-MS analysis. Two blank samples were prepared using the same procedures. A quality control (QC) sample was composed of 30 μL aliquots from each extracted WGC sample.

LC-MS analysis of metabolites

The detection conditions for the HPLC-MS system using the ACQUITY BEH Amide column (Waters, USA, 130 Å, 1.7 μm, 2.1 × 150 mm) are as follows: an injection volume of 2 μL, a column temperature of 50°C, and a flow rate of 0.3 mL/min. The mobile phase A is a 30% acetonitrile solution (containing 0.1% FA and 10 mM ammonium formate) and the mobile phase B is a 95% acetonitrile solution (containing 0.1% FA and 10 mM ammonium formate). Gradient elution: 0.0-1.0 min, 100% B, 1.1-11.0 min, 100%-30% B, 11.1-11.5 min, 30%-100% B, 11.6-15.0 min, 100% B. The mass spectrometry was performed using the HESI ionization spectrometry technique, with the mass spectrometer operating in positive and negative ion switching modes. Parameter settings: Ion source temperature is 320°C, with a spray voltage of 3.5 kV for positive ions and 3.0 kV for negative ions and parent ion scanning with a resolution of 70,000. The scanning range is 70-1000 m/z with an automatic gain control target ion number of 1×10⁶ and a maximum ion injection time of 50 ms.

Quantification and statistical analysis

To identify and quantify proteins, all MS/MS spectra were analyzed using Peaks Studio 8.5 (BSI, Canada),⁷⁰ against the sequences database generated from protein sequences of A. mellifera and common contaminants (downloaded April 2022), totaling 21,778 entries. The analysis utilized specific parameters, including a 20-ppm tolerance for precursor ions, a 0.05 Da tolerance for fragment ion mass, trypsin for enzymatic digestion with up to 2 missed cleavages, carbamidomethyl (C) as a constant modification, and oxidation (M) as a variable modification. Furthermore, stringent parameter settings were adopted for confident protein identification, requiring a false discovery rate (FDR) < 1% for peptides. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository⁷¹^,⁷² with the dataset identifier PXD047002.

To acquire a more profound comprehension of the biological functions of the proteins disclosed in the development of the WGC in honey bees, the Cytoscape (version 3.10.0) plugin ClueGO (version v2.5.10 ) was used to enrich for Gene Ontology (GO) terms.⁶⁶ By contrasting the input data with the background of GO categories in the honey bee genome and applying a hypergeometric right-sided test, it was able to find GO categories enriched in important biological processes. Networks of functionally grouped nodes were created using a kappa score of 0.4 as a basis for linking. Significance was determined based on a p-value threshold of < 0.05, with a Bonferroni step-down test used to control the false discovery rate. Then, the proteins were categorized according to their annotations to gain further insights into their specific functional roles. To investigate the statistically significant biological pathways associated with the identified proteins, we employed the KEGG Orthology-Based Annotation System⁷³ following the prescribed protocol.⁷⁴

Raw data files of metabolomics were imported into Compound Discoverer 3.2 (Thermo Fisher Scientific, USA) for ion peak identification, peak alignment, peak area normalization, and preliminary characterization using online databases including KEGG (KEGG: Kyoto Encyclopedia of Genes and Genomes) and HMDB (Human Metabolome Database) databases for compound identification.⁶⁵ To provide an overview of the relative abundance levels of the identified compounds, a heat map was generated with the use of ClustVis (Figure S1A).⁶⁷ Statistical and enrichment analyses were then performed using SIMCA 14.1 (Umetrics, Umea, Sweden). The overall metabolic differences of the samples between treatment groups were indicated by principal component analysis (Figure S1B). Orthogonal partial least squares discriminant analysis (OPLS-DA) reflected the magnitude of variability among samples between groups (Figure S1C). The OPLS-DA model was validated using permutation tests with 200 iterations (Figure S1D). The variable importance in projection (VIP) scores in the OPLS-DA was used to screen for compounds that contribute to the separation of the three groups. OPLS-DA plots were used to verify the stability of the model towards overfitting. R²X and R²Y represent the explanation rate of the model on the constructed X matrix and Y matrix, respectively. The predictive ability of the model is represented by Q², with Q² > 0.5 indicating good discriminant analysis ability. The proximity between Q² and R² signifies stability and the absence of overfitting. Differential metabolites were screened according to VIP score > 1, p-value < 0.05, |log₂FC| > 1, and finally enrichment analysis was performed.

Acknowledgments

This work is supported by the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2015-IAR), and the earmarked fund for Modern Agro-Industry Technology Research System (CARS-45) in China.

Author contributions

Y.F. and X.X. conceived the experiments; R.X. and B.M. performed study; Y.Y., X.D., and J.L. analyzed the data; R.X. and B.M. wrote the original manuscript; X.X. and Y. F. reviewed and revised.

Declaration of interests

The authors declare no competing interest.

Published: February 20, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.109279.

Contributor Information

Xiang Xu, Email: caasxx@163.com.

Yu Fang, Email: fangyu@caas.cn.

Supplemental information

Document S1. Figures S1 and S2

mmc1.pdf^{(500.8KB, pdf)}

Table S1. Identified proteins in WGC

The −10lgP value is a conversion of the p value, with a higher value indicating a more significant match. “#Peptides” indicates the total number of peptides assigned to the proteins, while “#Unique” represents the number of peptides exclusive to a single protein in the proteome. Sequence coverage is calculated as the percentage of amino acids in identified peptides compared to the total number of amino acids in the protein sequence.

mmc2.xls^{(780.5KB, xls)}

Table S2. Identified metabolic compounds in WGC

Raw data of metabolome were imported into Compound Discoverer 3.2 (Thermo Fisher Scientific) for ion peak identification, peak alignment, peak area normalization, and preliminary characterization using online databases including KEGG (Kyoto Encyclopedia of Genes and Genomes) and HMDB (Human Metabolome Database) databases for compound identification.

mmc3.xls^{(103.5KB, xls)}

Table S3. Identified three periods of common expression proteins (core proteins) and period-specific expression proteins in WGC

mmc4.xls^{(743.5KB, xls)}

Table S4. Biological pathway enrichment of core-expressed proteins identified at three developmental stages of the WGC

Significant biological pathway enrichment of the identified proteins was analyzed by the GO and KEGG plugins of ClueGO. Protein sequences were aligned with the A. mellifera database and then pathway enrichment was performed by hypergeometric statistical tests. Probability values were corrected using the Benjamini and Hochberg FDR correction, and only a corrected p < 0.05 was considered statistically significant for enriched biological pathways.

mmc5.xls^{(47.5KB, xls)}

Table S5. Analysis of core and specific metabolites of NEB-WSB and NEB-OAB

Metabolites all met the criteria of VIP >1, p < 0.05, and |log₂FC| > 1.

mmc6.xls^{(45.5KB, xls)}

Table S6. Biological pathway enrichment of specific expressed proteins identified in NEB, WSB, and OAB of the WGC

mmc7.xls^{(78.5KB, xls)}

Table S7. Expressional analysis of the differentially expressed significant (Fold change ≥1.5 and p < 0.05) proteins during the WGC development in honey bees

Label-free protein quantitation is done using the Peaks studio (version 8.5, Bioinformatics Solutions Inc., Waterloo, Canada) to quantify the abundance of discriminatory peptides based on retention time, m/z, and peak intensity (peak area) on samples. The differentially expressed proteins are considered to be statistically significant with p < 0.05 (using one-way ANOVA). The heatmap exhibits the correlations of expression trends over the developmental course of honeybee WGC by hierarchical clustering using an average linkage algorithm.

mmc8.xls^{(150KB, xls)}

Table S8. Biological pathway enrichment of up-regulated proteins identified in NEB, WSB, and OAB of the WGC

mmc9.xls^{(45.5KB, xls)}

Table S9. Identified compounds in WGC of WSB (NEB as control)

Metabolites were screened for compliance with Variable important in projection (VIP) > 1 through a multivariate analysis of model VIP values. Combination with p-value and fold change generated differential metabolites. 77 various substances were screened based on VIP >1 and |log₂FC| > 1 and p < 0.05. This process revealed 56 differential metabolites with up-regulated expression and 21 with down-regulated expression.

mmc10.xls^{(64KB, xls)}

Table S10. Identified compounds in WGC of OAB (NEB as control)

Metabolites were screened for compliance with Variable important in projection (VIP) > 1 through a multivariate analysis of model VIP values. Combination with p-value and fold change generated differential metabolites. 87 various substances were screened based on VIP >1 and |log₂FC| > 1 and p < 0.05. This process revealed 68 differential metabolites with up-regulated expression and 19 with down-regulated expression.

mmc11.xls^{(70KB, xls)}

Table S11. Enrichment analysis was conducted on the up-regulated expressed metabolites in both NEB-WSB and NEB-OAB groups using the oebiotech online cloud platform (https://www.oebiotech.com/index.php?c=show&id=414)

mmc12.xls^{(35.5KB, xls)}

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