Caste Differentiation in Apis Mellifera


In Apis Mellifera, commonly known as the European Honey Bee, there are different types of bees in the hive. There are foraging bees, which goes out and collects nectar, nursery bees which take care of the larvae and hive, and most famously queen bees. All these bees are of the same species yet there are clear physiological differences between a queen bee and a regular worker bee. A queen bee lives for years and can lay eggs while worker bees for the most part die off at the end of the summer.

What then causes larvae which are all born with the same set of genes to differentiate like they do? It doesn’t have to do with specific internal genes which are behind things like axis formation, but the inclusion of an outside protein called Royalactin induces differentiation between casts.

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All larvae when hatched are fed a diet of what is called royal jelly, food that worker bees have digested for consumption. After a couple of days, worker bees are weaned off the royal jelly but queen larvae are continually given royal jelly until maturation. Clearly then that royal jelly is the key to cast differentiation, but what in the jelly causes this? If we could know the answer then we could see what kind of health effects this substance could have and use it to better human health.

So far research has focused on a protein found in royal jelly called Royalactin (Rol).

To test this theory that Rol is behind the different phenotypes in bees, the researcher ran multiple experiments. Below is a summary of the three more interesting experiments ran.


The researchers first decided to look at how royal jelly degrades over time in honey bee larvae. What they found was that by 30 days, Rol had degraded to the point where it was no longer found in meaningful quantities. They then fed larvae a diet of either fresh royal jelly, degraded royal jelly, a diet consisting of 2% Rol, and finally a control substance.

Here we see the effects of all the different substances given to the larvae. Pay special attention to graph c as it shows the ovary size of the bees. If ovaries are small then they can’t lay eggs! Image taken from Kamakura(2011).


They found that the files with either fresh Royal jelly or with Rol were able to produce queen like phenotypes with a short digestion time, larger weight, and most importantly developed ovaries compared to degraded royal jelly or the control. Showing that Rol was a key ingredient in queen bee development and wasn’t depending on anything else in the royal jelly.


Then the researcher had the mad brilliant idea of giving the Rol to the fruit fly to see what affects the protein had on their development. a diet of pure royal jelly lead to the death of the fly larvae but a diet consisting of 20% royal jelly lead to larvae which survived. Once the researchers determined the ratio at which the larvae survived, they then gave the fly larvae either royal jelly, degraded royal jelly, a control food, degraded jelly with the control food, or degraded jelly with Rol added back in. What they found was that those flies who were fed the diet with Rol, no matter the source, were larger, heavier, and laid more eggs. Even more interestingly, the female flies of the Rol phenotype lived longer than the Rol-less flies, while there was no such difference in male flies.


Figure A shows a nice visual representation of the effects of Rol on flies. The graphs shows similar results where Rol increases size(B), number of eggs laid© and lifespan(D). An interesting take away is the Rol only affects lifespan of female flies, not male flies! Image taken from Kamakura(2011)


But what was Rol actually doing to the flies to cause such phenotypes?


Epidermal growth factor receptors(Egf) is a protein that functions similarly to Rol in mice. The researchers decided to investigate the relationship between Rol in Egf by raising fly larvae which had either an over-expression or underexpression of Rol and were also Egf mutants. These mutants were all raised on a non-royal jelly diet and measured on attributes like wait, life-span and number of eggs laid.


Instead of feeding the flies on a diet of Rol, here the author is overexpressing or underexposing the gene that produces Rol to see it’s effects. We see a similar patter to the previous experiment. Image taken from Kamakura(2011)


Flies with an over expression of Rol were shown to live longer, weigh more, and lay more eggs than their underexpressed counterparts. Interestingly, those over expressed Rol mutants who were also Egf mutants showed phenotypes closer to under-developed Rol mutants than over-expressed mutants.


Finally the researchers decided to use RNAi knockout to suppress Egf, GFP, InR, and S6K RNAi in honeybees then raised each mutants on a diet of Royal Jelly along with a control of a wild type larvae on degraded royal jelly.


The authors using RNAi knockout to see which gene interacts with Royal Jelly. GFP, InR, and S6K being top contenders. Image taken from Kamakura(2011)


Egf RNAi mutants were shown to be similar to the degraded royal jelly mutants while the other mutants showed no real difference.



…What this means is that Rol is a key ingredient in Royal Jelly which causes the jelly to overexpress the gene in queen larvae when fed enough. Rol is similar to Egf which is responsible for growth in cell size and other phenological differences that are found in queen bees compared to worker bees.


Kamakura, Masaki. “Royalactin induces queen differentiation in honeybees.” Nature 473.7348 (2011): 478-483.


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