Royal jelly is an important apiarian product for honeybees and has been used as an important ingredient to human health and healthy life style. Because of its wide use, there is great demand in their production. As royal jelly is a secretion of the cephalic glands of bees and it is produced at a certain age of the workers, it is necessary to perform the selection of producing queens to increase the amount produced. The employment of molecular markers is a tool that can be used to identify the genotypes of the best producers. Among the molecular markers, one of them called MRJP3 (Major Royal Jelly Protein 3) has been used in the Program of Improvement of Apis mellifera Royal Jelly Producing (PIAMRJP), State University of Maringá, Brazil. This molecular marker has been efficient in genotyping queens’ royal jelly producers. Combined with classical breeding studies, the selection of queens assisted by MRJP3 marker has allowed to keep the selected genotypes for royal jelly production since 2006 (10 years). In this chapter, we present the main aspects of royal jelly, the hypopharyngeal glands, the major proteins of royal jelly and how it can be used as molecular markers.
- Apis mellifera
- royal jelly
- honeybee queen
Genetic improvement in any organism has the objective of increasing the gene frequencies of the economic importance of loci to be selected in the population. In relation to bees, this means increasing the frequency of the number of colonies that produce above the average generation from which the selection was made.
The production of royal jelly and honey production are the result of the combined work of the workers , and therefore the entire colony becomes a unit of selection, where the assessment of improved queens is carried out by production workers’ progeny [1,2]. Royal jelly production studies allowed to observe considerable variation in its production by Africanized honeybees [3,4]. These results show the need for selection of queens .
Selection of bees with genotypes involves the use of improved queens’ replacement techniques and instrumental insemination and for molecular marker-assisted selection. There are few data in the literature linking molecular markers for the production of royal jelly. The identification and characterization of several loci of the major royal jelly proteins (MRJPs) allowed using one of the loci Mrjp3 as a molecular marker for selection of Apis mellifera queens Africanized. Early studies by selecting queens and genotyping the best producers began in 2006, in the apiary of the State University of Maringá, Brazil. The first study associating the MRJP3 marker with royal jelly production was realized by .
High variability in major royal jelly proteins (MRJPs), especially MRJP3 to contain microsatellite regions, indicated a great potential of using these proteins, particularly microsatellite regions occurring in the Mrjp3 gene as a marker for selecting studies for the improvement of the production of royal jelly. Subsequently, other researches were conducted using classic improvement parameters such as MRJP3 marker. The results to date have shown that this marker is important to genotype producing arrays of royal jelly.
Thus, this chapter shows the importance of royal jelly to honey bees and to human health, the importance of improving assisted by molecular markers and the results obtained with the selection of royal jelly producing queens and genotyped for MRJP3.
2. Royal jelly
Royal jelly is secreted by the mandibular and hypopharyngeal glands located at the head of honeybees . Hypopharyngeal gland secretion has a clear, water-like consistency and is rich in protein, while the mandibular gland produces a white secretion with milky consistency [7,8]. Royal jelly can be described as a viscous substance, white-yellowish or grayish white, slightly opalescent with a characteristic pungent odor, although not unpleasant or rancid (Figure 1) [9–10]. These glands have the highest growth rate and activity of worker nurses between days 10 and 14 [11–15]. The development of the glands can be influenced by internal factors of the colony such as offspring and population density and external factors such as foraging and enabling bees to adapt quickly to the colony [13,16–18].
Royal jelly is a glandular secretion recognized for complex composition, containing minerals, proteins, amino acids, steroids, phenols, carbohydrates, vitamins, lipids, acetylcholine and other unknown substances [9,19]; it is important too in reproduction and development. Royal jelly is the larval food until the third day of development when it becomes the exclusive food of the queen throughout her life, guaranteeing fertility and increased longevity. From third day, worker larvae are fed a mixture of honey, pollen and water, known as brood food; drones receive food brood and royal jelly .
The average lifespan of queens of A. mellifera live is 1 to 2 years ; they become sexually mature 6 days after emergence, mate about 17 drones and store all of the sperm needed to fertilize eggs for the duration of their lifespan . Few drones rear in the summer, but a slight rise in drone rearing occurs during swarming . Queens can lay 1500–2000 eggs per day throughout their lives [23,24], depending on the needs of the hive and environmental factors, while a large number of workers (sterile females) are responsible for maintaining the hive.
Due to the fertility and longevity of queens, related to the exclusive feeding with royal jelly, studies have been conducted considering similar effects in humans. Some beneficial effects have been attributed to consumption of royal jelly, as elimination of physical and mental fatigue, appetite normalization activation of brain function, improved vision, increased resistance against viral infections and skin rejuvenation .
Owing to considerable amount of proteins, free amino acids, lipids, vitamins, sugars and bioactive substances such as 10-hydroxy-trans-2-decenoic acid and antibacterial protein 350 KDa proteins, royal jelly becomes an ingredient for various healthy foods . Review carried out by  shows several studies have reported that the royal jelly exhibits beneficial physiological and pharmacological effects in mammals, including vasodilative and hypotensive activities, antihypercholesterolemic activity and antitumor activity.
3. Molecular marker Major Royal Jelly Protein 3
Royal jelly contains from 12 to 15% crude protein consisting of soluble proteins in water and water-insoluble proteins. The fraction of soluble proteins of royal jelly produced by the hypopharyngeal and mandibular glands contains several major proteins with molecular weight between 47 and 80 kDa  besides a small amount of minor proteins such as antibiotics and peptides [27,28]. Those proteins constitute the main group of major royal jelly proteins. The MRJPs represent between 82 and 90% of the total proteins of larval jelly . Some regions of MRJPs can be focused on amino acids rich in nitrogen, thus high levels of nitrogen would be stored in MRJPs. The availability of nitrogen can be critical to the rapid growth of young larvae, as well as for the development of the queen . These observations support the hypothesis that MRJPs have an important role in the nutrition of bees .
Major royal jelly protein-3 can be visualized on denaturing SDS-PAGE electrophoresis in head extracts of worker nurses (10–14 days old) or royal jelly (Figure 2). The polymorphism was estimated by .
MRJPs genes encoding a group of proteins that have a common evolutionary origin with Yellow proteins of Drosophila melanogaster . Genome of Drosophila encodes at least seven family members of Yellow proteins , whose loci are involved in the larval pigmentation , unlike the MRJPs that have nutritional function of larvae.
The genes encoding key proteins of royal jelly began to be identified in studies  and . After these pioneering studies, several studies have been published in order to identify and characterize new genes encoding the MRJPs proteins [19,31,36,37]. The availability of the complete genome of Apis mellifera  made it possible to identify new genes encoding proteins of the family MRJP .
Since the first study were identified nine proteins in MRJPs A. mellifera (MRJP1, MRJP2, MRJP3, MRJP4, MRJP5, MRJP6, MRJP7, MRJP8, and MRJP9) besides an incomplete polypeptide, MRJPψ, encoded by a pseudogene. The genes encoding these proteins are located on chromosome 11 . Classification of A. mellifera MRJPs has been performed based on the N-terminal sequences of purified protein and cDNA sequences available in the cDNA library.
Analysis using PCR and DNA sequencing showed that the different alleles of the gene encoding MRJP3 protein differ in length as a result of a varying number of repeating basic units in a region of the Mrjp3 gene [31,36]. The authors attributed the polymorphism of these proteins is a consequence of the presence of a region with varied number of repetitive sequences in tandem (microsatellite). These markers are comprised of a variable number of identical sequences having from 15 to 100 base pairs, in tandem and repeated up to 50 times. The molecular differences in four types of MRJP3 have shown that the polymorphism of these proteins is linked to the size variability, which is determined genetically by bees from the same colony .
The Mrjp3 is a polymorphic locus that has been identified by DNA sequencing five alleles and PCR analysis identified at least 10 alleles of different sizes . This study also revealed a Mendelian inheritance and high variability of the genomic locus of MRJP3.
Although A. mellifera [19,29,31,35–37] and other bees of the genus Apis [39–43] having the MRJPs are characterized, data in the literature on the use of MRJPs as molecular markers for selection associated with the improvement of royal jelly production are still scarce.
4. Apis mellifera queens’ selection using MRJP3 marker
The genetic improvement has the aim to increase the frequencies of desirable genes of the loci of economic importance to be selected in a population . Thus, the genetic breeding of bees has the goal to increase the frequency of the number of colonies that produce above the average generation from which the selection was made. Selection of honeybees with superior genotypes involves the use of improved queens replacement techniques, instrumental insemination and molecular marker-assisted selection .
Selection of queens is carried out by genetic evaluation, which depends on the estimation of the components of (co)variance and genetic parameters for identification of genetically higher bees. Royal jelly production evaluated by Bayesian inference had a heritability estimate of 0.27% acceptance, 0.10 for the production of royal jelly per colony and 0.55 per dome . The analyses performed by these authors showed that selection of queens can increase the production of royal jelly by colony, larval acceptance and production of royal jelly by the dome, and the external factors can modify the gene expression of individuals.
However, there are few data in the literature associating molecular markers for the production of royal jelly. One of the first molecular studies carried out to obtain DNA markers related to production of royal jelly was performed by . These authors reviewed a total of 96 alleles produced for 10 microsatellite loci and according to the observed allele frequency for some alleles, it was possible to identify seven alleles that can be used as markers bees producing large quantities of royal jelly. The use of molecular markers, particularly microsatellites, can contribute to detect polymorphisms that might be useful to identify colonies of bees with high productivity of royal jelly.
High variability of MRJPs proteins and especially the MRJP3 to contain microsatellite region shows a great potential to use MRJP family proteins as markers for selection of producing queens for improving the production of royal jelly. Use of MRJPs as molecular markers in studies of population genetics and as selection markers associated with the improvement of royal jelly production is still scarce. Some researches have shown that this molecular marker is efficient to be used in the selection of royal jelly-producing queens.
Africanized honeybees selected for royal jelly production showed high allelic variability for the locus Mrjp3 (Figure 3), showing the potential of this marker for selection . In this research analyses of multiple linear regressions with EPD (expected progeny differences) values for royal jelly production were performed. The variance analyses indicated that the Mrjp3 repetitive region influenced the genetic value of queen’s offspring for royal jelly production. The determination coefficient (R2) for the significant alleles of the repetitive region of Mrjp3 indicated that 36.85% of the EPD variation is explained by the variation of C, D and E alleles. Authors concluded that the three alleles present a considerable genetic effect on the variation of royal jelly production.
Continuing the process of selection and the Program of Improvement of Apis mellifera Royal Jelly Producing (PIAMRJP), State University of Maringá, Brazil, alleles of the locus Mrjp3 descendants queens, those selected by , were evaluated in 2011 . Results showed that the royal jelly-producing queens had a high degree of genetic diversity and excess homozygous alleles. The highest frequencies were estimated for Mrjp3 D and E alleles 0.3357 and 0.3107, respectively, showing that the selection process of queens royal jelly producing these alleles are being maintained and only the C allele had a low frequency of 0.0321.
Results obtained by  confirm those obtained by , the locus Mrjp3 and their alleles C, D and E influence the genetic value for producing royal jelly; however, the real role of MRJP3 these bees has not yet been identified. The sequencing of Mrjp3 of A. mellifera Africanized alleles in PIAMRJP was performed . Homology and identity of these sequences were compared with the sequences deposited in the database for A. mellifera (Figure 4). Alleles Mrjp3 detected showed high identities with alleles deposited in BLAST system. Alleles Mrjp3 C, D and E are being maintained in the genome of the selected matrices queens.
High similarity among the Mrjp3 alleles analyzed and those described in other studies show that the Mrjp3 locus is conserved among species and subspecies of Apis. Similar results were obtained by . These authors found that there are high similarity sequences and intron-exon have the same structure between four species A. mellifera, A. cerana, A. dorsata and A. florea.
The selection of royal jelly-producing queens may be promoting a selection of these reproduction bees, can alter the genetic characteristics of a given population, can be influenced by the process of transmission of these genes generation to generation . However, it is important to maintain a degree of genetic variation, which results in a larger potential response to selective improvement .
In addition to the continuous genotyping of royal jelly–producing queens to locus Mrjp3, we developed a study to see if the mitochondrial DNA (mtDNA) of Africanized bees A. mellifera maintained in the breeding program have African or European origin. This research was performed by , using matrices producing royal jelly.
Mitochondrial DNA was analyzed using the molecular marker PCR-RFLP with specific primers and restriction enzymes to European and African honey bees. Analyses were performed with workers’ daughters of royal jelly-producing queens in 2013, seven years after the beginning of the PIAMRJP started in 2006. After this period of selection and analysis of genetic parameters, alleles C, D and E are being maintained in queens, evidencing the role in royal jelly production. Queens selected for royal jelly production showed predominance of African mtDNA; therefore, genes of maternal origin are African. Use of microsatellite markers and mtDNA can be used in bee improvement programs to ensure the genetic origin of queens and verify the efficiency of Program of Improvement of Apis mellifera Royal Jelly Producing .
The employment of molecular markers in selection programs and improvement of honey bees for royal jelly production is efficient because it allows keeping genotypes of interest to ensure the highest productivity of the hives. The microsatellite marker MRJP3 has shown good results as a tool to verify the genotypes of producing matrices, facilitating identification and maintenance of the hives in the apiary of the Program of Improvement of Apis mellifera Royal Jelly Producing.