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
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
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
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
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
Since the first study were identified nine proteins in MRJPs
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
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
Continuing the process of selection and the Program of Improvement of
Results obtained by  confirm those obtained by , the locus
High similarity among the
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
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
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
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