Proteomics analysis reveals protein expression differences for hypopharyngeal gland activity in the honeybee, Apis mellifera carnica Pollmann

Abstract

Background: Most of the proteins contained in royal jelly (RJ) are secreted from the hypopharyngeal glands (HG) of young bees. Although generic protein composition of RJ has been investigated, little is known about how age-dependent changes on HG secretion affect RJ composition and their biological consequences. In this study, we identified differentially expressed proteins (DEPs) during HG development by using the isobaric tag for relative and absolute quantification (iTRAQ) labeling technique. This proteomic method increases the potential for new protein discovery by improving the identification of low quantity proteins.

Results: A total of 1282 proteins were identified from five age groups of worker bees, 284 of which were differentially expressed. 43 (15.1%) of the DEPs were identified for the first time. Comparison of samples at day 6, 9, 12, and 16 of development relative to day 3 led to the unambiguous identification of 112, 117, 127, and 127 DEPs, respectively. The majority of these DEPs were up-regulated in the older worker groups, indicating a substantial change in the pattern of proteins expressed after 3 days. DEPs were identified among all the age groups, suggesting that changes in protein expression during HG ontogeny are concomitant with different states of worker development. A total of 649 proteins were mapped to canonical signaling pathways found in the Kyoto Encyclopedia of Genes and Genomes (KEGG), which were preferentially associated with metabolism and biosynthesis of secondary metabolites. More than 10 key high-abundance proteins were involved in signaling pathways related to ribosome function and protein processing in the endoplasmic reticulum. The results were validated by qPCR.

Conclusion: Our approach demonstrates that HG experienced important changes in protein expression during its ontogenic development, which supports the secretion of proteins involved in diverse functions in adult workers beyond its traditional role in royal jelly production.

Figure 1

DEPs distribution and expression levels in samples. (A) The x-axis shows the pairwise comparisons of the five sample groups (days 3, 6, 9, 12, and 16), and the y-axis displays the number of proteins. Red and green bars indicate up-regulated and down-regulated proteins, respectively. (B) Venn analysis of DEPs in the five samples (Adapted from Chen et al. [48]). The numbers denote the amount of proteins that were expressed in each class, with arrows indicating the number of up-regulated (↑) or down-regulated (↓) proteins. Classes are labeled from a to o based on the following representations: (a) exclusively expressed in group d6 vs. d3; (b) exclusively expressed in group d9 vs. d3; (c) exclusively expressed in group d12 vs. d3; (d) exclusively expressed in group d16 vs. d3; (e) only expressed in group d6 vs. d3 and d9 vs. d3; (f) only expressed in group d6 vs. d3 and d12 vs. d3; (g) only expressed in group d6 vs. d3 and d16 vs. d3; (h) only expressed in group d9 vs. d3 and d12 vs. d3; (i) only expressed in group d9 vs. d3 and d16 vs. d3; (j) only expressed in group d12 vs. d3 and d16 vs. d3; (k) exclusively not expressed in group d16 vs. d3; (l) exclusively not expressed in group d12 vs. d3; (m) exclusively not expressed in group d9 vs. d3; (n) exclusively not expressed in group d6 vs. d3; (o) expressed in common. (C) Expression profiling of DEPs common among all the samples. The first 7 proteins were down-regulated with log2-transformed fold-change ratios that were no more than 0.5. The other proteins were up-regulated by no less than 1.5-fold. Note: It should be noted that all the redundant proteins were included in the Venn analysis.

Figure 2

Classification of GO categories. The three functional categories were (A) cellular component, (B) biological process, and (C) molecular function. (D) WEGO output of our data. Hierarchical GO tree in which all the GO terms contained in the plot are shown to compare the annotation results. x-axis indicates functional items. y-axis (left) shows the percent of the proteins. y-axis (right) represents the number of proteins.

Figure 3

Distribution of DEP abundances. (A-D) DEPs at specific time points were relative to day 3 as the baseline. The x-axis indicates the fold-change (ratios) of proteins based on the logarithm with base 2. The y-axis indicates the protein ID. The candidate DEPs were indicated in red (up-regulated) or green (down-regulated), with the absolute value of log₂ (protein fold-change) >1.5. The range of log-fold changes in panel B and C were much wider than panel A and D, revealed more DEPs change their expression pattern in d9 and d12 compared with d3 and d16. That may show us the right direction to focus their functions in the regulation of HG activity.

Figure 4

GO enrichment analysis of DEPs. Panel A to D represent the compared groups of day 6 vs day 3, day 9 vs. d3, day 12 vs. d3 and day 16 vs. d3. To the left of each plot: GO terms. Above each plot: -log₁₀ (p-value). Yellow bars indicate cellular component, red bars represent a biological process, and blue bars denote a molecular function.

Figure 5

Hierarchical Clustering of multiple samples (56 proteins in all). Each column represents and compares a group relative to day 3, (1: d6 vs. d3; 2: d9 vs. d3; 3: d12 vs. d3; 4: d16 vs. d3) and each row represents a gene. Expression differences are shown in different colors; red indicates up-regulation, whereas green indicates down-regulation.

Figure 6

Correlation between the expression levels of 35 proteins and their corresponding gene transcripts. (A) The expression levels of proteins and their transcripts in d16 vs. d3 were used for a whole understanding to reveal the correlation between the two methodologies, showing the significant positive correlation (r = 0.3828, P = 0.010). (B) The qPCR results show the better correlation with r = 0.496, P = 0.002. The fold-change (ratios) of proteins and transcripts were based on the logarithm with base 2.