Royalactin induces queen differentiation in honeybees


The honeybee (Apis mellifera) forms two female castes: the queen and the worker. This dimorphism depends not on genetic differences, but on ingestion of royal jelly, although the mechanism through which royal jelly regulates caste differentiation has long remained unknown. Here I show that a 57-kDa protein in royal jelly, previously designated as royalactin, induces the differentiation of honeybee larvae into queens. Royalactin increased body size and ovary development and shortened developmental time in honeybees. Surprisingly, it also showed similar effects in the fruitfly (Drosophila melanogaster). Mechanistic studies revealed that royalactin activated p70 S6 kinase, which was responsible for the increase of body size, increased the activity of mitogen-activated protein kinase, which was involved in the decreased developmental time, and increased the titre of juvenile hormone, an essential hormone for ovary development. Knockdown of epidermal growth factor receptor (Egfr) expression in the fat body of honeybees and fruitflies resulted in a defect of all phenotypes induced by royalactin, showing that Egfr mediates these actions. These findings indicate that a specific factor in royal jelly, royalactin, drives queen development through an Egfr-mediated signalling pathway.

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Figure 1: Effects of casein, 450-kDa protein, royalactin and recombinant royalactin on caste characters in the honeybee.
Figure 2: Morphological and physiological changes of Drosophila melanogaster induced by royal jelly and royalactin.
Figure 3: Morphological and physiological changes of Drosophila melanogaster induced by overexpression of royalactin.
Figure 4: Suppression of queen differentiation in honeybee with RNAi.

Change history

  • 25 May 2011

    The legend to Fig. 1 was corrected


  1. 1

    Maynard Smith, J. & Szathmary, L. The Major Transitions in Evolution (Freeman, 1995).

    Google Scholar 

  2. 2

    Haydak, M. H. Honey bee nutrition. Annu. Rev. Entomol. 15, 143–156 (1970)

    Article  Google Scholar 

  3. 3

    Patel, N. G., Haydak, M. H. & Gochnauer, T. A. Electrophoretic components of the proteins in honeybee larval food. Nature 186, 633–634 (1960)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Weaver, N. Effects of larval age on dimorphic differentiation of the female honey bee. Ann. Entomol. Soc. Am. 50, 283–294 (1957)

    Article  Google Scholar 

  5. 5

    Shuel, R. W. & Dixon, S. E. The early establishment of dimorphism in the female honeybee, Apis mellifera L. Insectes Soc. 7, 265–282 (1960)

    Article  Google Scholar 

  6. 6

    Page, R. E. & Peng, C. Y. Aging and development in social insects with emphasis on the honey bee, Apis mellifera L. Exp. Gerontol. 36, 695–711 (2001)

    Article  Google Scholar 

  7. 7

    Wheeler, D. E. Developmental and physiological determinations of caste in social Hymenoptera: evolutionary implications. Am. Nat. 128, 13–34 (1986)

    Article  Google Scholar 

  8. 8

    Patel, A. et al. The making of a queen: TOR pathway is a key player in diphenic caste development. PLoS ONE 2, e509 (2007)

    ADS  Article  Google Scholar 

  9. 9

    Kamakura, M., Mitani, N., Fukuda, T. & Fukushima, M. Antifatigue effect of fresh royal jelly in mice. J. Nutr. Sci. Vitaminol. (Tokyo) 47, 394–401 (2001)

    CAS  Article  Google Scholar 

  10. 10

    Beetsma, J. The process of queen-worker differentiation in the honeybee. Bee World 60, 24–39 (1979)

    CAS  Article  Google Scholar 

  11. 11

    Kamakura, M., Suenobu, N. & Fukushima, M. 57-kDa protein in royal jelly enhances proliferation of primary cultured rat hepatocytes and increases albumin production in the absence of serum. Biochem. Biophys. Res. Commun. 282, 865–874 (2001)

    CAS  Article  Google Scholar 

  12. 12

    Kamakura, M. Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-kDa protein. J. Biochem. 132, 911–919 (2002)

    CAS  Article  Google Scholar 

  13. 13

    Bloch, G., Wheeler, D. E. & Robinson, G. E. in Hormones, Brain and Behavior Vol. 3 (eds Pfaff, D. W., Arnold, A. P., Fahrbach, S. E., Etgen, A. M. & Rubin, R. T. ) 195–235 (Academic Press, 2002)

    Google Scholar 

  14. 14

    Wirtz, P. & Beetsma, J. Induction of caste differentiation in the honeybee (Apis mellifera L.) by juvenile hormone. Entomol. Exp. Appl. 15, 517–520 (1972)

    CAS  Article  Google Scholar 

  15. 15

    Tatar, M. et al. A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 292, 107–110 (2001)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Colombani, J. et al. Antagonistic actions of ecdysone and insulins determine final size in Drosophila . Science 310, 667–670 (2005)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Gilboa, L. & Lehmann, R. Soma-germline interactions coordinate homeostasis and growth in the Drosophila gonad. Nature 443, 97–100 (2006)

    ADS  CAS  Article  Google Scholar 

  18. 18

    Caldwell, P. E., Walkiewicz, M. & Stern, M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release. Curr. Biol. 15, 1785–1795 (2005)

    CAS  Article  Google Scholar 

  19. 19

    Mirth, C., Truman, J. W. & Riddiford, L. M. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster . Curr. Biol. 15, 1796–1807 (2005)

    CAS  Article  Google Scholar 

  20. 20

    Belgacem, Y. H. & Martin, J. R. Hmgcr in the Corpus Allatum controls sexual dimorphism of locomotor activity and body size via the insulin pathway in Drosophila . PLoS ONE 2, e187 (2007)

    ADS  Article  Google Scholar 

  21. 21

    Colombani, J. et al. A nutrient sensor mechanism controls Drosophila growth. Cell 114, 739–749 (2003)

    CAS  Article  Google Scholar 

  22. 22

    Zinke, I. et al. Suppression of food intake and growth by amino acids in Drosophila: the role of pumpless, a fat body expressed gene with homology to vertebrate glycine cleavage system. Development 126, 5275–5284 (1999)

    CAS  PubMed  Google Scholar 

  23. 23

    Navolanic, P. M., Steelman, L. S. & McCubrey, J. A. EGFR family signaling and its association with breast cancer development and resistance to chemotherapy. Int. J. Oncol. 22, 237–252 (2003)

    CAS  PubMed  Google Scholar 

  24. 24

    LeVea, C. M., Reeder, J. E. & Mooney, R. A. EGF-dependent cell cycle progression is controlled by density-dependent regulation of Akt activation. Exp. Cell Res. 297, 272–284 (2004)

    CAS  Article  Google Scholar 

  25. 25

    Rintelen, F., Stocker, H., Thomas, G. & Hafen, E. PDK1 regulates growth through Akt and S6K in Drosophila . Proc. Natl Acad. Sci. USA 98, 15020–15025 (2001)

    ADS  CAS  Article  Google Scholar 

  26. 26

    McManus, E. J. & Alessi, D. R. TSC1–TSC2: a complex tale of PKB-mediated S6K regulation. Nature Cell Biol. 4, E214–E216 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Lee, K. S. et al. Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling. Nature Cell Biol. 10, 468–475 (2008)

    CAS  Article  Google Scholar 

  28. 28

    Montagne, J. et al. Drosophila S6 kinase: a regulator of cell size. Science 285, 2126–2129 (1999)

    CAS  Article  Google Scholar 

  29. 29

    Zhang, H. et al. Regulation of cellular growth by the Drosophila target of rapamycin dTOR . Genes Dev. 14, 2712–2724 (2000)

    CAS  Article  Google Scholar 

  30. 30

    Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993)

    CAS  Google Scholar 

  31. 31

    Urban, S. Rhomboid proteins: conserved membrane proteases with divergent biological functions. Genes Dev. 20, 3054–3068 (2006)

    CAS  Article  Google Scholar 

  32. 32

    Schmitzova, J. et al. A family of royal jelly proteins of the honeybee. Apis mellifera L. Cell. Mol. Life Sci. 54, 1020–1030 (1998)

    CAS  Article  Google Scholar 

  33. 33

    Bownes, M. The regulation of the yolk protein genes, a family of sex differentiation genes in Drosophila melanogaster . Bioessays 16, 745–752 (1994)

    CAS  Article  Google Scholar 

  34. 34

    Goewie, E. A. & Beetsma, J. Induction of caste differentiation in the honey bee (Apis mellifera L.) after topical application of JH-III. Proc. K. Ned. Akad. Wet. C 79, 466–469 (1976)

    Google Scholar 

  35. 35

    Ebert, R. Influence of juvenile hormone on gravity orientation in the female honeybee larvae (Apis mellifera L.). J. Comp. Physiol. 137, 7–16 (1980)

    CAS  Article  Google Scholar 

  36. 36

    Bíliková, K. et al. Apisimin, a new serine-valine-rich peptide from honeybee (Apis mellifera L.) royal jelly: purification and molecular characterization. FEBS Lett. 528, 125–129 (2002)

    Article  Google Scholar 

  37. 37

    Kamakura, M. & Sakaki, T. A hypopharyngeal gland protein of the worker honeybee Apis mellifera L. enhances proliferation of primary-cultured rat hepatocytes and suppresses apoptosis in the absence of serum. Protein Expr. Purif. 45, 307–314 (2006)

    CAS  Article  Google Scholar 

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I thank D. Yamamoto for provision of general fruitfly treatment methods and helpful advice; and S. Hayashi and T. Adachi-Yamada for instruction of dissection techniques in Drosophila. I thank T. Nonogaki and Y. Hasada for supply of honeybee larvae; K. Yu, M. Tatar, P. Leopold, G. Korge, Y. T. Ip, T. G. Wilson and D. Yamamoto for fly stocks. We are grateful to T. Oda for the gift of royal jelly, and to W. R. S. Steele for proofreading the article.

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M.K. designed the research and performed the experiments. M.K. wrote the paper.

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Correspondence to Masaki Kamakura.

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The author declares no competing financial interests.

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Supplementary Information

This file contains Supplementary Results, a Supplementary Discussion, Supplementary Methods, additional references, Supplementary Figures 1-34 with legends and Supplementary Tables 1-14. This file was replaced online on 15 October 2012 to correct Supplementary Figures 22a and 33c and again on 23 January 2013 to correct Supplementary Figure 19b.. (PDF 4372 kb)

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Kamakura, M. Royalactin induces queen differentiation in honeybees. Nature 473, 478–483 (2011).

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