The Foraging Time Analysis
The duration of honeybees imbibing food in imidacloprid group and deltamethrin group was significantly lower than the control group (p < 0.05). The returning period was significantly higher in imidacloprid group and deltamethrin group compared with the control group (p < 0.05) (Fig.1).
Differentially Expressed Genes Detected in all Samples
A total of 85 DEGs were identified in the imidacloprid-treatment group versus the control group, and 122 DEGs were identified in the deltamethrin-treatment group versus the control group. Additionally, 27 DEGs were shared in both treatment groups, including apidaecin, hymenoptaecin, CYP 450 6a14, CYP450 303a1, TpnCI, TpnCIIb, Mrjp1, calmodulin-like protein and other uncharacterized proteins (Fig.2). There were 58 DEGs specific to the imidacloprid-treatment group and 95 DEGs were specific to the deltamethrin-treatment group (Fig.2). In the imidacloprid-treatment group, 60 genes were up-regulated, including 10 detoxification immune genes, 6 metabolization-related genes, 6 sensory and signal transduction genes, and 38 other genes. There were 25 down-regulated differential genes, including 2 involved with detoxification and immunity, 1 metabolism-related gene, 2 for sensory and signaling, and 20 for other genes (Fig.3). In the deltamethrin-treatment group, 76 genes were up-regulated, 10 for detoxification and immunity, 7 for metabolization-related genes, 6 for sensory and signal transduction, and 53 for other genes. There were 46 down-regulated differentially expressed genes, including 5 detoxification genes, 2 metabolically related genes, 11 sensory and signaling genes, and 28 other genes (Fig.3).
Interestingly, 35 genes of the DEGs between insecticide-treated and control bees were involved in detoxification, immunity, sensation and signal transduction. There were 17 genes with significant differences in the imidacloprid-treatment group and 25 genes in the deltamethrin-treatment group (Table S1, Fig.4). There were eight commonly up-regulated genes in both groups, including cytochrome P450 6a14, peroxisomal multifunctional enzyme type 2-like, apidaecins type 73, hymenoptaecin preproprotein, troponin C type I, troponin C type Iib and calmodulin-like protein 4. The significantly down-regulated gene was major royal jelly protein 1 in both groups.
GO Enrichment Analysis
In the imidacloprid-treatment group and deltamethrin-treatment group, the DEGs were significantly enriched in 7 categories of biological function, namely response to biotic stimulus (GO: 0009607), response to bacterium (GO: 0009617), response to external biotic stimulus (GO: 0043207), response to other organisms (GO: 0051707), glucose metabolic process (GO: 0006006), defense response (GO: 0006952) and response to external stimuli (GO: 0009605). These categories are closely related to biological activities such as metabolism, defense response, chemical sensing and response to external stimuli.
Furthermore, three categories in deltamethrin group reached significant enrichment level (adjusted P value < 0.05) for molecular functions, including nucleotide binding (GO: 0000166), oxidoreductase activity (GO: 0016491) and nucleoside phosphate binding (GO: 1901265). In addition, biological function analysis also found ten significant enrichment terms. These categories are related to biological activities such as cytoskeleton, signal transduction, receptor activity, ion balance, defense response, metabolism, and biosynthesis processes.
Pathway Analysis of DEGs
Fifteen DEGs were enriched into 32 pathways in the imidacloprid group compared with the control group. Three pathways reached significantly enrichment, including Lysine biosynthesis (ko00300), Lysine degradation (ko00310) and Phosphatidylinositol signaling system (ko04070) (Table 1). These pathways are mainly involved in metabolism, amino acid metabolism, drug metabolism, detoxification and immunity. Eighteen DEGs were enriched to 35 pathways in the deltamethrin group compared with the control group. Three pathways reached significantly enrichment, namely Glycine, serine and threonine metabolism (ko00260), Metabolic pathways (ko01100) and Hippo signaling pathway-fly (ko04391) (Table 1). These pathways are mainly related to biological activities such as metabolism, amino acid metabolism, signal transduction, detoxification, drug metabolism and endocytosis.
Table 1
Pathway enrichment analysis for the significantly expressed genes in imidacloprid-treated bees and deltamethrin-treated bees.
Control vs Imidacloprid Pathway Enrichment
|
Control vs Deltamethrin Pathway Enrichment
|
Pathway ID
|
Pathway
|
Differentially expressed genes
|
P-value
|
Pathway ID
|
Pathway
|
Differentially expressed genes
|
P-value
|
ko00300
|
Lysine biosynthesis
|
724239
|
0.017
|
ko00260
|
Glycine, serine and threonine metabolism
|
551044, 552457
|
0.020
|
ko00310
|
Lysine degradation
|
102654572,
724239
|
0.020
|
ko01100
|
tMetabolic pathways
|
102653682, 102655219, 412843, 551044, 551726, 552457, 725017, 725215, 727387, 727584
|
0.023
|
ko04070
|
Phosphatidylinositol signaling system
|
551726
|
0.031
|
ko04391
|
Hippo signaling pathway-fly
|
552637, 727368
|
0.048
|