Comparative 4D Label-Free Quantitative Proteomic Analysis of Bombus terrestris Provides Insights into Proteins and Processes Associated with Diapause

Abstract

Diapause, an adaptative strategy for survival under harsh conditions, is a dynamic multi-stage process. Bombus terrestris, an important agricultural pollinator, is declining in the wild, but artificial breeding is possible by imitating natural conditions. Mated queen bees enter reproductive diapause in winter and recover in spring, but the regulatory mechanisms remain unclear. Herein, we conducted a comparative 4D label-free proteomic analysis of queen bees during artificial breeding at seven timepoints, including pre-diapause, diapause, and post-diapause stages. Through bioinformatics analysis of proteomic and detection of substance content changes, our results found that, during pre-diapause stages, queen bees had active mitochondria with high levels of oxidative phosphorylation, high body weight, and glycogen and TAG content, all of which support energy consumption during subsequent diapause. During diapause stages, body weight and water content were decreased but glycerol increased, contributing to cold resistance. Dopamine content, immune defense, and protein phosphorylation were elevated, while fat metabolism, protein export, cell communication, signal transduction, and hydrolase activity decreased. Following diapause termination, JH titer, water, fatty acid, and pyruvate levels increased, catabolism, synaptic transmission, and insulin signaling were stimulated, ribosome and cell cycle proteins were upregulated, and cell proliferation was accelerated. Meanwhile, TAG and glycogen content decreased, and ovaries gradually developed. These findings illuminate changes occurring in queen bees at different diapause stages during commercial production.

Keywords: 4D label-free quantitative proteomics; Bombus terrestris; diapause; post-diapause; pre-diapause; protein abundance.

Figure 1

Overall proteomic analyses of Bombus terrestris queens during adult reproductive diapause. (A) Basic proteome information. (B) Distribution of protein sequence coverage; the numbers in the pie chart represent the number of proteins with the same color of sequence coverage. (C) tSNE plot of samples (n = 3 for each treatment). The upset plot shows the numbers and intersections of identified proteins (D), the top 50 most abundant proteins (E), and DAPs for six group comparisons (F) from different B. terrestris proteomes. (G) Histogram of upregulated (red) and downregulated (blue) DAPs proteins for six group comparisons.

Figure 2

KEGG pathway enrichment analyses of DAPs of Bombus terrestris in each comparison group. (A) BD vs. M. (B) D1W vs. BD. (C) D12W vs. D1W. (D) BDT vs. D12W. (E) PD 48 h vs. BDT. (F) OPD vs. PD 48 h. Red represents upregulated proteins, blue represents downregulated proteins, and numbers in diagrams represent DAP numbers.

Figure 3

Clustering of protein expression profiles of Bombus terrestris using a fuzzy c-means algorithm. (A) Expression patterns of four clusters generated by Mfuzz software. (B) KEGG enrichment analysis of the four clusters. GO enrichment analysis of (C) molecular function, (D) biological process, and (E) cell component subcategories.

Figure 4

Heatmap of proteins involved in fat metabolism in Bombus terrestris. Results are presented as means of three independent samples. Scale bar shows upregulation (red) and downregulation (blue). The magnitude of the regulation is illustrated by the intensity of the color, and the number on the scale bar represents log2 (mean relative quantitative value).

Figure 5

Heatmap of genes and proteins involved in glycolysis (A) and the TCA cycle (B) in Bombus terrestris. Results are presented as means of three independent samples. Scale bar shows upregulation (red) and downregulation (blue). The magnitude of the regulation is illustrated by the intensity of the color, and the number on the scale bar represents log2 (mean relative quantitative value).

Figure 6

Substance content determination of Bombus terrestris for fresh weight (A), dry weight (B), water content (C), total lipids (D), TAG (E), FFAs (F), ovary phenotypes (G), glycogen (H), proteins (I), pyruvate (J), trehalose (K), glycerol (L), dopamine (M), insulin (N), JH titers (O), and ecdysone (P).

Figure 7

Summary of protein expression profiles and altered substances during pre-diapause, diapause, and post-diapause in Bombus terrestris. Red arrows represent upregulation, and the green arrows represent downregulation.