Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (11): 2464-2475.doi: 10.3864/j.issn.0578-1752.2021.11.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

The Capping Pheromone Contents and Putative Biosynthetic Pathways in Larvae of Honeybees Apis cernana

QIN QiuHong1,2(),HE XuJiang1,JIANG WuJun3,WANG ZiLong1,ZENG ZhiJiang1()   

  1. 1Honeybee Research Institute, Jiangxi Agricultural University/Jiangxi Province Key Laboratory of Honeybee Biology and Beekeeping, Nanchang 330045
    2School of Medicine, Guangxi University of Science and Technology, Liuzhou 545005, Guangxi
    3Apicultural Research Institute of Jiangxi Province, Nanchang 330052
  • Received:2020-08-24 Accepted:2020-11-09 Online:2021-06-01 Published:2021-06-09
  • Contact: ZhiJiang ZENG E-mail:qqhpcc87@163.com;bees1965@sina.com

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Abstract:

【Objective】 In honeybees, methyl palmitate (MP), methyl oleate (MO), methyl linoleate (ML) and methyl linolenate (MLN) are important capping pheromone components, which trigger the capping behavior of adult workers. The objective of this study is to compare the contents of these four pheromone components in the larvae of workers and drones of Apis cernana at different capping stages, analyze their biosynthetic pathways, and to further explore the mechanism of pheromone communication between larvae and adult workers. 【Method】Using A. c. cernana as the experimental material, the larvae of workers and drones of prior to be capped, in the process of being capped and had been capped were collected for comparing the contents of these four pheromone components by using GC/MS. Simultaneously, RNA-seq was used for gene expression analysis, and the biosynthetic pathways were speculated based on KEGG enrichment of differential expressed genes. 【Result】In worker larvae, the contents of the four capping pheromone components were significantly higher at the capping and capped stage than those of the prior to be capped larvae, and the contents of MP and MO significantly increased with aging of the larvae, while the contents of ML and MLN were not significantly different between the capping and capped stage. Whereas in drone larvae, the contents of the four pheromone components were higher overall and increased with aging, and the content at capped stage was significantly higher than that at prior to be capped and capping. RNA-seq results showed that there were 4 299 and 3 926 differential expressed genes among the larvae groups of three stages of workers and drones, respectively. In addition, 152 and 130 KEGG pathways were obtained from the KEGG annotation analysis of the differential expressed genes, respectively. Furthermore, the possible de novo biosynthetic pathways were proposed for MP, MO, ML and MLN from acetyl-CoA, regulating under 11 related candidate genes, and these biosynthesis pathways were found to be similar to those of Apis mellifera. 【Conclusion】The release contents of MP, MO, ML and MLN were increased during the critical stage of capping in worker and drone larvae of A. cernana, which further verified that these four pheromones were related to capping behavior of honeybees, and it was speculated that they were possibly de novo biosynthesized from acetyl-CoA under the control of related genes. A. cernana larvae and A. mellifera larvae may use the same biosynthesis pathway for pheromone biosynthesis.

Key words: Apis cernana, capping pheromone, content, transcriptome, biosynthetic pathway

Table 1

qRT-PCR primers"

基因编号
Gene ID		 基因名称
Gene name		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
gene-APICC_00421 Kat CCACGGCCTCAAACGACTC CGGCACCATCAGAAATACCAG
gene-APICC_06057 Hadha GGAGGAGGCTTGGAGATGG GCACCAGGCAAGATACCTAACA
gene-APICC_00389 Mecr TGCCTTCACCAAAATTAGCCC CCATGCTGTCATCCAAAATCC
gene-APICC_01118 Δ11 desaturase GCTTGTCAATTCCGCTGCTC AACCTTCGCCAAGTGCTCCTA
gene-APICC_02088 Hacd GCAGCTTCATCTTTTGCTCTTC CTATAACTCCATGCTTCTGGGTG
β-actin TCCTGGAATCGCAGATAGAATG GGAAGGTGGACAAAGAAGCAAG

Fig. 1

The contents of capping pheromone extracted from worker larvae at different capping stages The data in the figure were mean±S.E. Different letters on the bars indicated significant differences (P<0.05). The same as Fig. 2"

Fig. 2

The contents of capping pheromone extracted from drone larvae at different capping stages"

Fig. 3

The number of DEGs in worker and drone larvae of different capping stages"

Table 2

Annotation of candidate genes related to the biosynthesis of capping pheromones in larvae of workers and drones"

基因编号Gene ID 基因注释Gene annotation
gene-APICC_00421 3-ketoacyl-CoA thiolase (Apis cerana cerana)
gene-APICC_06057 Trifunctional enzyme subunit alpha (Apis cerana cerana)
gene-APICC_10032 Enoyl-CoA hydratase (Apis cerana cerana)
gene-APICC_00389 PREDICTED: probable trans-2-enoyl-CoA reductase, mitochondrial (Apis cerana)
gene-APICC_05477 PREDICTED: elongation of very long chain fatty acids protein 4-like isoform X1 (Apis dorsata)
gene-APICC_02088 PREDICTED: very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase hpo-8 (Apis cerana)
gene-APICC_05451 Trans-2,3-enoyl-CoA reductase (Apis cerana cerana)
gene-APICC_01118 Acyl-CoA Delta (11) desaturase (Apis cerana cerana)
gene-APICC_08222 Acyl-CoA desaturase (Apis cerana cerana)
gene-APICC_01590 PREDICTED: acyl-CoA Delta (11) desaturase-like (Apis cerana)
gene-APICC_03413 Acyl-CoA desaturase (Apis cerana cerana)

Fig. 4

Proposed biosynthetic pathways involved in the synthesis of capping pheromone of A. cerana, and expression of related genes in worker and drone larvae of different capping stages The grids illustrate the gene expression levels for different capping stages larvae of workers and drones. Grey scale density of each cell shows the absolute expression magnitude of honeybee larvae, with the FPKM values 0-10, 10-20, 20-40, 40-80, 80-160, 160-320, 320-640, 640-1280, 1280-2560 and 2560-5500 represented by gray scale levels, respectively"

Fig. 5

Expression levels of five genes possibly implicated in the biosynthesis of capping pheromone components in larvae of different capping stages The data in the figure were mean±S.E. Different letters on the bars indicated significant differences (qRT-PCR: P<0.05; RNA-seq: FDR≤0.05)"

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