*3.1. RJ Enhances Fertility and Longevity in Population of the Beehive*

Numerous bioactive elements are abundant in RJ, making it an optimal food and a well-balanced nutrient-rich diet [25] that, when consumed by bee larvae, induces their development into queens. Meanwhile, larvae that consume honey or pollen grow into workers, which live shorter and are unable to reproduce. On the other hand, emerging bee workers fed RJ-rich diet acquire queen-like morphogenic characteristics such as increased body size and ovary development [41,60,61]. Real-time RT-PCR and HPLC-ECD analysis denoted that augmentation of fertility (ovarian development) in bee workers by RJ is the result of enhancement of brain levels of tyrosine, dopamine, and tyramine by RJ [61], as well as activation of epidermal growth factor receptor (*EGFR*), which increases the production of the juvenile hormone, titre, known to regulate growth [41]. RJ also improves the memory of bee workers [62] and increases their survival [15,41,56,62,63], owing to its high Ach concentration [56], MRJP1 content of royalactin [41], MRJPs 2, 3, and 5 [25], and a water soluble RJ protein extracted by precipitation with 60% ammonium sulfate (RJP60) [15].

#### *3.2. RJ Enhances Healthspan and Longevity in Other Species*

Based on the assumption that RJ is the main factor contributing to long survival of bee queens, several studies examined the lifespan-expanding effects of RJ in diverse species (Figure 2), and results seem to be consistent with the naturally occurring model of bee queens.

**Figure 2.** Royal jelly and its components improve healthspan and extend lifespan in different species.

#### 3.2.1. Drosophila Melanogaster (Fruit Fly)

The fruit fly *Drosophila melanogaster* (*M.*) has been widely used in the literature as an invertebrate model to understand the pathology of numerous diseases and to examine the effect of various agents, including RJ. The earliest study of the longevity effect of RJ dates back to 1948. That study reported that large doses of dehydrated RJ, its pantothenic acid, as well as its water soluble and insoluble organic acids significantly extended lifespan of *Drosophila M.* compared with controls. The author suggested that pantothenic acid of RJ may have an anti-aging effect by itself or by synergizing the action of other vitamins [54]. In a relatively recent study, RJ (0.1, 0.3, and 0.5 g) significantly increased the average lifespan of both male and female flies—the effect was attributable to enhancement of the antioxidation capacity noticed in flies treated by high doses of RJ: increased superoxide dismutase (SOD) and catalase levels [64].

As a replication of his experiment of RJ and royalactin in bees, Kamakura (2011) treated *Drosophila Canton-S.* larvae with royalactin and 20% fresh RJ. RJ treatment increased body size, cell size, and fertility; prolonged lifespan; and shortened developmental time from larva to adult compared with control. Same as in bees, RT-PCR and enzyme immunoassay showed that both RJ and royalactin induced *EGFR* signaling (not insulin signaling), which activated *S6K* in the fat body, which further increased the body size. *EGFR* also activated the *MAPK* pathway, which increased the synthesis of a biologically active ecdysteroid known as 20-hydroxyecdysone (*20E*) and juvenile hormone—an effect that was demonstrated by growth regulation—reducing the developmental time [41]. A subsequent study stated that supplementing *Drosophila M.* with 1% freeze-dried RJ (FDRJ) powder shortened developmental time, prolonged the lifespan of adult males, and increased females' egg production without any morphological changes. In female flies, RT-PCR indicated that FDRJ was significantly associated with heightened gene expression of an insulin-like peptide known as dilp5, its insulin receptor (*InR*), and the nutrient sensing molecule *mTOR*, the mechanistic target of rapamycin—all these molecules are known to affect growth and reproduction. However, stimulation of the *insulin*/*TOR* signaling pathways was not associated with extension of lifespan of FDRJ-fed female flies [17]. Another study noted that supplementation of MRJPs, especially MRJP 1 and MRJP3, at an optimal dose of 2.5% (*w*/*w*) of diet significantly lengthened the mean lifespan of both male and female *Drosophila*. The longevity effect of MRJPs was positively associated with increased feeding and fertility. Microarray data and gene ontology enrichment analyses revealed that the molecular mechanism underlying increased lifespan and fertility was similar to that discovered by Kamakura (2011): MRJP supplementation upregulated the gene expression of *S6K*, *MAPK*, and *EGFR* in *EGFR*-mediated signaling. In addition, MRJPs improved the anti-oxidation capacity of flies by increasing the expression of *CuZn-SOD* gene

i.e., SOD levels were higher, while malonaldehyde (MDA) levels were lower than control flies [12]. Another study replicated the size/growth enhancing effect of RJ at low concentrations (10–30%) in *Drosophila M.* [16]. However, males' lifespan decreased by 20% RJ treatment whereas none of these concentrations affected the lifespan of females. Findings provided no evidence of RJ activation of insulin and *EGFR* signaling pathways, yet RJ regulated the gene expression related to oxidative stress and catabolism. The authors attributed the discrepancy noted between their results and findings of other studies to employing a slightly different strain of *Drosophila* (*Canton-S*), using a commercially available source of RJ, and difference in the nutrient contents of the control culture medium. On the other hand, higher concentrations (40–70%) of RJ had adverse effects: prolonged development time, shortened lifespan, increased mortality, and reduced productivity in both sexes. Data on global gene expression indicated that excess nutrients in high doses of RJ altered cellular processes as a result of altering genes involved in amino acid metabolism and encoding glutathione S transferases, which detoxify xenobiotic compounds [16].
