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.
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About this article
Nature Communications (2018)
Communications Biology (2018)
Longevity extension of worker honey bees (Apis mellifera) by royal jelly: optimal dose and active ingredient