The food jelly of honey bees

Honey bees do not feed their larvae directly with the collected nectar or pollen, but the young honey bee workers (also called nurse bees) produce foraging sap from it (Swammerdam, 1738). This occurs in the hypopharyngeal and mandibular glands, which are located in the head of the workers (Kratky, 1931; Schiemenz, 1883). Feeding juice is therefore a glandular secretion. All honey bee larvae get first of all basically this feed juice (von Planta, 1888; 1889); but depending on whether the larva then develops into a worker, queen or drone, the composition of the food sap differs slightly; above all, the amount of food sap that the different larvae receive differs (Snodgrass, 1925; von Planta, 1888, 1889; Wang et al., 2016). Feeding juice fed to queen larvae is also called royal jelly (Huber, 1792).

Food jelly contains all kinds of nutrients that honey bee larvae need, including proteins that are synthesised and secreted by the glandular cells of the hypopharyngeal gland (Patel et al., 1960). In the early 1990s, the main protein from royal jelly was isolated and named major royal jelly protein (MRJP) (Hanes & Šimúth, 1992). However, royal jelly does not only contain this one protein (see figure) and over time it was discovered that almost all of these food jelly proteins have a similar amino acid sequence and thus belong to one protein family, the major royal jelly protein (MRJP) family. Due to the high occurrence of essential amino acids in these proteins, it was previously assumed that the proteins mainly have a nutritional function during larval development (Albert et al., 1999a, 1999b; Schmitzová et al., 1998).

With the publication of the genome of the western honey bee, it was then clear that Apis mellifera has a total of nine genes that are responsible for MRJPs encode, and the genes were simply numbered from one to nine (Weinstock et al., 2006). The main royal jelly protein, first isolated in 1992, has been assigned number one and is now called MRJP1. Interestingly, all nine mrjp genes in succession on the same chromosome and probably arose by gene duplication (Drapeau et al., 2006). All other bee species sequenced so far, e.g. the dark earth bumblebee Bombus terrestris and the alfalfa leafcutter bee Megachile rotundata, have only one MRJP (Kapheim et al., 2015; Sadd et al., 2015).

Now, as you can easily guess after this introduction, my research revolves around these MRJPs. I am particularly interested in the functions of the nine MRJPs in Apis mellifera , why other bee species do only have a single MRJP and what the function of this single MRJP is.

The complex of MRJP1 and apisimin

Although all nine MRJPs can be detected in royal jelly using mass spectrometry (Schönleben et al., 2007; Zhang et al., 2014), MRJP1, 2 and 3 make up the majority of proteins in royal jelly (Schmitzová et al., 1998). When MRJP1 is purified from royal jelly, it is often co-purified with the relatively small protein apisimin (approx. 5 kDa) (Bíliková et al., 2002). This is because MRJP1 and apisimin bind to each other and form a protein complex (Mandacaru et al., 2017; Tamura et al., 2009;Tian et al., 2018). The function of this complex was previously unknown. We could now show that the complex, which at a neutral pH of 7.0 consists of four molecules of MRJP1 and four molecules of apisimin (Mandacaru et al., 2017; Tian et al., 2018), starts to form fibrillar structures at a pH below 5.0 (Buttstedt et al., 2018). At pH 4.0, the natural pH of royal jelly, the two proteins start to form beautiful protein fibrils. In addition, we were able to show via electron microscopy that royal jelly is composed of many fibrillar structures, which are destroyed when the pH is artificially raised to pH 7.0 (Kurth et al., 2019).

Now, two questions arose: What is the function of these protein fibrils? And is the acidic pH of royal jelly actually important?

And of course it is! 🙂 At a natural pH of 4.0, royal jelly has a viscosity about three times higher than at an artificially raised pH of over 5.0 (Buttstedt et al, 2018). This means that if the pH is too high, royal jelly becomes more liquid. And this would have fatal consequences for the queen larvae (see video), as they hang from the ceiling in their queen cells and are stuck to the ceiling with the royal jelly. Thus, MRJP1 and apisimin in the form of the fibrils in the royal jelly ensure the survival of the royal offspring. The low pH of royal jelly is adjusted by adding the secretion from the mandibular gland to the protein-containing secretion from the hypopharyngeal gland. This secretion contains many free fatty acids, especially 10-hydroxy-2-decenoic acid (10-HDA), and the lowering of the pH of the feeding juice caused by this fatty acid causes the fibril formation of the complex (Buttstedt, 2022).

Another advantage of low royal jelly pH is that MRJP1, 2, and 3 are more heat stable at acidic pH.

Another advantage of the low pH of royal jelly is that MRJP1, 2 and 3 are more stable at acidic pH(Mureşan & Buttstedt, 2019) and significantly more resistant to degradation by proteases (Mureşan et al., 2018) than at neutral pH.

Another component of the complex of MRJP1 and apisimin is the plant sterol 24-methylenecholesterol (Tian et al, 2018). While the proteins MRJP1 and Apisimin, as mentioned above, originate from the hypopharyngeal gland, 24-methylenecholesterol is found together with the fatty acids in the mandibular gland secretion and is thus added to the feeding juice (Buttstedt et al., 2023). This means that both the protein complex and the fibrils are formed only after the hypopharyngeal and mandibular gland secretions are mixed together.