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  • Brief Communications Arising
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Royalactin is not a royal making of a queen

Nature volume 537, pages E10–E12 (2016)Cite this article

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ARISING FROM M. Kamakura Nature 473, 478–483 (2011)

Honeybee (Apis mellifera) females occur in two castes: workers and one reproductive queen. Caste is nutritionally regulated and only larvae exclusively fed on royal jelly (RJ) develop into queens. Decades of search for a queen ‘determinator’ in RJ found no specific compound, concluding that the discrete feeding regime throughout larval development controlled caste fate. Thus, Kamakura’s claim1 that the monomeric major royal jelly protein 1 (MRJP1, ‘royalactin’), induces queen differentiation was surprising in light of previous research1. Because mechanisms of caste determination are crucial to social insect research, we revisited the topic and show that this is incorrect by experimentally excluding not only monomeric MRJP1 but also any other major royal jelly protein (MRJP2, 3 and 5) as single key driver for queen caste determination. There is a Reply to this Brief Communication Arising by Kamakura, M. Nature 537, http://dx.doi.org/10.1038/nature19350 (2016).

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Figure 1: Analysis and purification of monomeric MRJP1.
Figure 2: Frequency distribution of castes in adult bees reared on different diets.

References

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Authors and Affiliations

  1. Institut für Biologie, Zoologie – Molekulare Ökologie, Martin-Luther-Universität Halle-Wittenberg, Hoher Weg 4, Halle (Saale), 06120, Germany

    Anja Buttstedt, Markus Pietzsch & Robin F. A. Moritz

  2. Institut für Pharmazie – Aufarbeitung biotechnischer Produkte, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, Halle (Saale), 06120, Germany

    Anja Buttstedt

  3. Institut für Pharmazie – Pharmazeutische Chemie & Bioanalytik, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4, Halle (Saale), 06120, Germany

    Christian H. Ihling

  4. Department of Zoology and Entomology, University of Pretoria, Pretoria, 0002, South Africa

    Robin F. A. Moritz

Authors
  1. Anja Buttstedt
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  2. Christian H. Ihling
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  3. Markus Pietzsch
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  4. Robin F. A. Moritz
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Corresponding author

Correspondence to Anja Buttstedt.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 In vitro rearing of larvae and queen determination rates among different studies.

a, In vitro rearing of honeybee larvae. b, Queen determination rates of in vitro rearing trials among different studies and diets. An additional increase of sugar content in the normal RJ diet (12% (w/w) glucose and fructose, each) slightly, but not significantly, increased queen rate (5.9%). Our queen rates are well in agreement with a median queen determination rate of 9.4% (0–28.3%) in all other published in vitro larvae rearing experiments using 50 to 66% RJ diets. Although there is considerable variance in the queen determination rate within and among different laboratories, bee genotypes and probably RJ sources, it has never, to our knowledge, approached 100%.

Extended Data Figure 2 Phenotypic scoring.

a, Mandible categories. b, Tibia categories. c, Spermatheca diameters of virgin queens, in vitro bees and hive-reared workers. d, Ovary diameters of virgin queens, in vitro bees and hive-reared workers. e, Stinger angles of virgin queens, in vitro bees and hive-reared workers. f, Parameters quantified in workers, queens and in vitro-raised honeybees.

Extended Data Figure 3 Analysis of MRJP1 via gel-filtration and mass spectrometry.

a, Gel-filtration analysis (HiLoad 16/600 Superdex 200 pg column) of MRJP1 present in the flow through (FT) (blue line) and eluting with 300 mM NaCl (red line) from the cation exchange chromatography (SP sepharose). In 20 mM sodium citrate pH 4.0, 150 mM NaCl (upper panel) MRJP1 is present as higher oligomers (FT fraction) and as monomer (300 mM NaCl elution fraction). In 50 mM potassium phosphate pH 7.0, 150 mM NaCl, the higher oligomers detected in the FT fraction dissociate mainly into hexamer (~90%) and minor portions of dodecamer (~5%), monomer (~4%) and probably trimer (~1%). Monomeric MRJP1 detected in the citrate buffer is in phosphate buffer mostly present as monomer (~91%) but also as hexamer (~9%). The different elution volumes of monomeric MRJP1 in pH 4.0 (84 ml = 57 kDa) and pH 7.0 (82 ml = 43 kDa) may result from different hydrodynamic volumes of the protein in the respective buffers. As the hydrodynamic volume appears to change largely between pH 4.0 and 7.0, this suggests that the structure of the monomer is different at the different pH values. The column was calibrated with ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa) (dashed black line) and the void volume determined with blue dextran (2,000 kDa) (solid black line). b, MRJP1 has been identified with 68.8% sequence coverage (red amino acids) by mass spectrometry. Mass deviation between measured and theoretical masses was not exceeding 5 p.p.m. and 0.02 Da for precursor and fragment ions, respectively.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Table 2 (see separate file for Supplementary Table 1) and additional references. (PDF 292 kb)

Supplementary Tables

This file contains Supplementary Table 1 comprising a complete list with details for all measured parameters for all 538 examined bees (Sheet 1) and for all Kruskal-Wallis ANOVAs (Sheet 2) and Chi-square tests (Sheet 3). (XLSX 87 kb)

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Buttstedt, A., Ihling, C., Pietzsch, M. et al. Royalactin is not a royal making of a queen. Nature 537, E10–E12 (2016). https://doi.org/10.1038/nature19349

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