Kamakura replies

An Erratum to this article was published on 09 November 2016

REPLYING TO A. Buttstedt, C. H. Ihling, M. Pietzsch & R. F. A. Moritz Nature 537, 10.1038/nature19349 (2016)

In the accompanying Comment1, Buttstedt et al. investigated the effects of royalactin on queen differentiation in honeybees, and claimed that there is no effect of royalactin on queen development, contrary to my findings2. However, the experimental conditions in Buttstedt et al. do not appear to be adequate to induce queen honeybee differentiation.

First, with ImageJ, I quantified the intensity of the royalactin band (lane RJ) in fig. 1a of Buttstedt et al. using the stable oligomeric MRJP1 as a standard: it was only 21% of that in their fresh royal jelly (RJ) sample (lane FRJ). This suggests that the royalactin concentration in RJ used in the experiments could be lower than that in the fresh RJ. Furthermore, using the purification method (with SP sepharose) described by Buttstedt et al., I detected monomeric MRJP1 as doublet bands in the elution fraction with 300 mM NaCl, which was detected with a gel-filtration column as a single peak (Supplementary Fig. 1a, lane 2). Hence, royalactin purified by Buttstedt et al. does not seem to be pure enough and sufficient for the in vitro rearing system.

Secondary, Buttstedt et al. used a general larvae-rearing method established to examine the pathogens and the toxicity of compounds3,4, which was different from my rearing method2. Especially, they did not give the larvae sufficient food to induce queen development. Buttstedt et al. fed the larvae with 300 μl (355 mg) of the diet containing 50% of RJ per well and day, which is only 177.5 mg of RJ. Hence, for rearing queen larvae, the amount of RJ in their diet was much less than the requisite amount (239 mg) of 100% RJ5. In addition, this amount of RJ diet medium in each well of a 24-well plate in their experiment (300 μl) results in a depth of only 0.8–1.0 mm. From day 4, which is the crucial time point for queen development6, to day 7, the height of larvae reared with my rearing method is 4.0–6.0 mm, or even more (Supplementary Fig. 1b). As the larval mouth is at the centreline of their body, the depth of the diet should be more than 3.0 mm for the induction of queens. These results indicate that the larvae in the experiments of Buttstedt et al. could not eat a sufficient amount of royalactin for queen development. By contrast, to ensure their sufficient uptake of the food, I used 1,000 μl of RJ diet (1,085 mg) containing 50% RJ (542.5 mg) per well per day with a depth of 3.5–4.0 mm (ref. 2). It is known that larval weights, adult weights and adult ovariole number are reduced when larvae are fed in vitro with limited food7,8. Indeed, I confirmed that the feeding method used by Buttstedt et al. (300 μl per well) results in poor larval growth with lower weight, which would prevent larva becoming queens (Supplementary Fig. 1c)5,7,8,9. The concentration of royalactin in RJ used by Buttstedt et al. was 3.0 mg g−1, while that in my RJ was 21.0 mg g−1 (refs 2,10). Therefore, the amount of royalactin in 300 μl of the RJ diet used by Buttstedt et al. per well was 0.53 mg per well, while in my experiments, 1,000 μl of my RJ diet per well equates to 11.4 mg of royalactin per well. These results indicate that larvae were not fed with sufficient RJ and royalactin to induce queens in the experiments of Buttstedt et al.

Finally, the experiments presented in Buttstedt et al. have no positive control. Buttstedt et al. also did not examine their fresh RJ. I argue that larvae could not develop into queen honeybees under the inappropriate conditions described above. My administration test of royalactin and genetic analyses using Drosophila melanogaster also support my claim that royalactin induced caste differentiation in honeybees2.


  1. 1

    Buttstedt, A., Ihling, C. H., Pietzsch, M. & Moritz, R. F. A. Royalactin is not a royal making of a queen. Nature 537, http://dx.doi.org/10.1038nature19349 (2016)

  2. 2

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

    CAS  ADS  Article  Google Scholar 

  3. 3

    Crailsheim, K. et al. Standard methods for artificial rearing of Apis mellifera larvae. J. Apic. Res. 52, 1–16 (2013)

    Article  Google Scholar 

  4. 4

    Genersch, E., Ashiralieva, A. & Fries, I. Strain- and genotype-specific differences in virulence of Paenibacillus larvae subsp. larvae, a bacterial pathogen causing American foulbrood disease in honeybees. Appl. Environ. Microbiol. 71, 7551–7555 (2005)

    CAS  Article  Google Scholar 

  5. 5

    Asencot, M. & Lensky, Y. The phagostimulatory effect of sugars on the induction of “queenliness” in female honeybee (Apis mellifera L.) larvae. Comp. Biochem. Physiol. 81A, 203–208 (1985)

    Article  Google Scholar 

  6. 6

    Wang, D. I. & Shuel, R. W. Studies in the mode of action of royal jelly in honeybee development V. the influence of diet on ovary development. J. Apic. Res. 4, 149–160 (1965)

    Article  Google Scholar 

  7. 7

    Kaftanoglu, O., Linksvayer, T. A. & Page, R. E. Rearing honey bees (Apis mellifera L.) in vitro: effects of feeding intervals on survival and development. J. Apic. Res. 49, 311–317 (2010)

    Article  Google Scholar 

  8. 8

    Linksvayer, T. A. et al. Larval and nurse worker control of developmental plasticity and the evolution of honey bee queen-worker dimorphism. J. Evol. Biol. 24, 1939–1948 (2011)

  9. 9

    Asencot, M. & Lensky, Y. The effect of soluble sugars in stored royal jelly on the differentiation of female honey bee (Apis mellifera L.) larvae to queens. Insect Biochem. 18, 127–133 (1988)

    CAS  Article  Google Scholar 

  10. 10

    Kamakura, M., Fukuda, T., Fukushima, M. & Yonekura, M. Storage-dependent degradation of 57-kDa protein in royal jelly: a possible marker for freshness. Biosci. Biotechnol. Biochem. 65, 277–284 (2001)

    CAS  Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to M. Kamakura.

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1 and additional references. This file was replaced on 20th October 2016 to correct the formatting of the figure legend. (PDF 232 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kamakura, M. Kamakura replies. Nature 537, E13 (2016). https://doi.org/10.1038/nature19350

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing