Clarence Collison
Caste Determination
The capacity of the honey bee to produce three phenotypically distinct organisms (two female castes; queens and sterile workers, and haploid male drones) from one genotype represents one of the most remarkable examples of developmental plasticity. The queen-worker morphological and reproductive divide is environmentally controlled during post-embryonic development by differential feeding. Previous studies implicated metabolic flux (rate of turnover of molecules through a metabolic pathway) acting via epigenetic regulation (changes in organisms caused by modification of gene expression rather than alteration of the genetic code), in particular DNA methylation and microRNAs, in establishing distinct patterns of gene expression underlying caste-specific developmental trajectories. The first genome-wide maps of chromatin structure in the honey bee were produced at a key larval stage in which developmental canalization into queen or worker is virtually irreversible. Extensive genome-wide differences were found in H3K4me3, H3K27ac and H3K36me3, many of which correlate with caste-specific transcription. Transcription is the process of copying a segment of DNA into RNA molecules. Genetic information flows from DNA into protein, the substance that gives an organism its form. The flow of information occurs through the sequential processes of transcription (DNA to RNA) and translation (RNA to protein). Furthermore, H3K27ac was identified as a key chromatin modification, with caste-specific regions of intronic H3K27ac directing the worker caste. These regions may harbor the first examples of caste-specific enhancer elements in the honey bee. These results demonstrate a key role for chromatin modifications in the establishment and maintenance of caste-specific transcriptional programs in the honey bee. It was shown that at 96 hours of larval growth, the queen-specific chromatin pattern is already established, whereas the worker determination is not, thus providing experimental support for the perceived timing of this critical point in developmental heterochrony in two types of honey bee females (Wojciechowski et al. 2018).
Artificial queen rearing with worker larvae grafted at different developmental stages resulted in gradual effects on ovary size (number of ovarioles per ovary), as well as hind leg and wax gland structures in adults. A significant decrease in ovariole number was observed when third instar larvae were grafted. Basitarsus shape was affected when fourth instar larvae were grafted. Queen—worker intermediates developed when early-fifth instar worker larvae were transferred. As newly emerged adults, spectra of cephalic (of, in or relating to the head) volatiles (pheromones) of queens and workers are still very similar, and do not yet exhibit the caste-specific elements of the mandibular glands. At one day after emergence, most of the dominant compounds in these spectra are represented at higher levels in workers (Dedej et al. 1998).
Female honey bees have two castes: queens and workers. Developmental fate is determined by larval diet. Coding sequences made available through the Honey Bee Genome Sequencing Consortium allow for a pathway-based approach to understanding caste determination. Wheeler et al. (2006) examined the expression of several genes of the insulin signaling pathway, which is central to regulation of growth based on nutrition. They found one insulin-like peptide expressed at very high levels in queen, but not worker, larvae. Also, the gene for an insulin receptor was expressed at higher levels in queen larvae during the second larval instar. These results demonstrate that the insulin pathway is a compelling candidate for pursuing the relationship between diet and downstream signals involved in caste determination and differentiation.
Caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. Illumina/Solexa sequencing was employed to examine the microRNAs (abbreviated miRNA) content in the larval food. They found that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were seven to 215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the fourth to sixth day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval miRNA expression and morphological characters of the emerging adult queen bee. They proposed that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee (Guo et al. 2013).
A female larva’s developmental fate depends on its diet; nurse bees feed queen-destined larvae exclusively royal jelly, a glandular secretion, but worker-destined larvae receive royal jelly for three days and subsequently jelly to which honey and beebread are added. RNA-Seq analysis demonstrated that p-coumaric acid, which is ubiquitous in honey and beebread, differentially regulates genes involved in caste determination. Rearing larvae in vitro on a royal jelly diet to which p-coumaric acid has been added produces adults with reduced ovary development. Thus, consuming royal jelly exclusively not only enriches the diet of queen-destined larvae but also may protect them from inhibitory effects of phytochemicals present in the honey and beebread fed to worker-destined larvae (Mao et al. 2015).
The development of queen and worker castes is induced by differential nutrition, with future queens and workers receiving diets that are qualitatively and quantitatively different. Wheeler et al. (2014) monitored the gene expression of 14 genes for components of the insulin/insulin-like signaling and TOR pathways in honey bee larvae from 40-88 hours after hatching. They compared normally fed queen and normally fed worker larvae and found that three genes showed expression differences in 40 hour old larvae. Genes that show such early differences in expression may be part of the mechanism that transduces nutrition level into a hormone signal. They then compared changes in expression after shifts in diet with those in normally developing queens and workers. Following a shift to the worker diet, the expression of nine out of 14 genes was upregulated in comparison with queens. Following a shift to the queen diet, expression of only one gene changed. The honey bee responses may function together as a homeostatic mechanism buffering larvae from caste-disrupting variation in nutrition. The different responses would be part of the canalization of both the queen and worker developmental pathways.
Honey bees use differential feeding and a haplodiploid sex determination system to generate three distinct organismal outcomes from the same genome. Ashby et al. (2016) investigated the honey bee female and male caste-specific microRNA and transcriptomic molecular signatures during a critical time of larval development. Both previously undetected and novel miRNAs have been discovered, expanding the inventory of these genomic regulators in invertebrates. Significant differences in the microRNA and transcriptional profiles of diploid females relative to haploid drone males as well as between reproductively distinct females (queens and workers) were shown. Queens and drones show gene enrichment in physio-metabolic pathways, whereas workers show enrichment in processes associated with neuronal development, cell signaling and caste biased structural differences. Interestingly, predicted miRNA targets are primarily associated with non-physio-metabolic genes, especially neuronal targets, suggesting a mechanistic disjunction from DNA methylation that regulates physio-metabolic processes. Accordingly, miRNA targets are under-represented in methylated genes. Their data show how a common set of genetic elements are differentially harnessed by an organism, which may provide the remarkable level of developmental flexibility required.
The development of a larva into either a queen or worker depends on differential feeding with royal jelly and involves epigenomic modifications by DNA methyltransferases. To understand the role of DNA methylation in this process, Foret et al. (2012) sequenced the larval methylomes in both queens and workers. They showed that the number of differentially methylated genes (DMGs) in larval head is significantly increased relative to adult brain (2,399 vs. 560) with more than 80% of DMGs up-methylated in worker larvae. Several highly conserved metabolic and signaling pathways are enriched in methylated genes, underscoring the connection between dietary intake and metabolic flux. This includes genes related to juvenile hormone and insulin, two hormones shown previously to regulate caste determination.
Regardless of genetic makeup, a female honey bee becomes a queen or worker depending on the food she receives as a larva. For decades, it has been known that nutrition and juvenile hormone (JH) signaling determine the caste fate of the individual bee. However, it is still largely unclear how these factors are connected. To address this question, Mutti et al. (2011) suppressed nutrient sensing by RNA interference (RNAi)-mediated gene knockdown of IRS (insulin receptor substrate) and TOR (target of rapamycin) in larvae reared on queen diet. The treatments affected several layers of organismal organization that could play a role in the response to differential nutrition between castes. These include transcript profiles, proteomic patterns, lipid levels, DNA methylation response and morphological features. Most importantly, gene knockdown abolished a JH peak that signals queen development and resulted in a worker phenotype. Application of JH rescued the queen phenotype in either knockdown, which demonstrates that the larval response to JH remains intact and can drive normal developmental plasticity even when IRS or TOR transcript levels are reduced. They found that IRS is an alternative substrate for Egfr (epidermal growth factor receptor) in honey bees. Overall, their study describes how the interplay of nutritional and hormonal signals affects many levels of organismal organization to build different phenotypes from identical genotypes.
Corpora-allata (produces juvenile hormone) activity of queen and worker larvae of the honey bee in late larval development was studied in vitro by a radiochemical assay. Prospective queens showed a high peak of corpora allata activity in the fourth and early fifth larval stadium. This peak coincides with a queen-specific maximum in juvenile hormone titre, demonstrating that modulation of juvenile hormone release is of prime importance in the regulation of the caste-specific juvenile hormone titre. In both castes, hormone release is strictly correlated with juvenile hormone synthesis. The conversion of the precursor methyl farnesoate to juvenile hormone may be regulated caste-specifically, since only in queens but not in workers, a linear correlation between intraglandular contents of juvenile hormone and methyl farnesoate could be found (Rachinsky and Hartfelder 1990).
Queens and workers are alternative forms of the adult female honey bee and represent one of the best known examples of insect polyphenism (two or more distinct phenotypes are produced by the same genotype). Hormonal regulation of caste determination in honey bees has been studied in detail, but little is known about the proximate molecular mechanisms underlying this process, or any other such polyphenism. They report the success of a molecular-genetic approach for studying queen and worker specific gene expression in the development of the honey bee. Numerous genes appear to be differentially expressed between the two castes. Seven differentially expressed loci described here belong to at least five distinctly different evolutionary and functional groups. Two are particularly promising as potential regulators of caste differentiation. One is homologous to a widespread class of proteins that bind lipids and other hydrophobic ligands, including retinoic acid. The second locus shows sequence similarity to a DNA binding domain in the Ets family of transcription factors. The remaining loci appear to be involved with downstream changes inherent to queen or worker specific developmental pathways. Caste determination in honey bees is typically thought of as primarily queen determination; the results make it clear that the process involves specific activation of genes in workers as well as in queens (Evans and Wheeler 1999).
Newly emerged honey bee larvae were reared in the laboratory on 32P-labeled royal jelly. The resulting adults were classified as workers, intermediates or queens, depending on their morphological caste characters. Larvae destined to become queens ate 13% more food than worker larvae during the first three days of larval life. This difference increased to about 40% after six days of larval life. The mean rate of ingestion was 8% less in intermediates as compared to queen larvae during the first three days after hatching, and 16% less after six days of larval life. Queen larvae consumed an average of 5% more royal jelly than intermediates, and 19% more than workers. The results are discussed in relation to the known differences in growth rates of the queen-worker castes (Dietz and Lambremont 1970).
Honey bee larval growth is markedly influenced by the moisture content of the larval food. A gradual increase in the moisture content of the larval food (royal jelly) resulted in a large percentage of queens. An apparent linear relationship of royal jelly moisture content to time of larval development was used in the feeding trials. Since the larval food tested was one and one-half or two and one-half years old, it is probable that a highly labile queen determining substance, or substances, if present in larval food, is not responsible for caste determination. Results of this study suggest that nurse bees may initiate the mechanism of queen differentiation simply by increasing the moisture content of the food of growing larvae and thereby, controlling the intake of essential nutrients. The secretions of the mandibular glands are presumably responsible for the change in consistency of the food of either queen or worker larvae (Dietz and Haydak 1971).
References
Ashby, R., S. Forêt, I. Searle, and R. Maleszka 2016. MicroRNAs in honey bee caste determination. Sci. Rep. 6:18794, doi:10.1038/srep18794
Dedej, S., K. Hartfelder, P. Aumeier, P. Rozenkranz, and W. Engels 1998. Caste determination is a sequential process: effect of larval age at grafting on ovariole number, hind leg size and cephalic volatiles in the honey bee (Apis mellifera carnica). J. Apic. Res. 37: 183-190.
Dietz, A. and M.H. Haydak 1971. Caste determination in honey bees. I. The significance of moisture in larval food. J. Exp. Zool. 177: 353-357.
Dietz, A. and E.N. Lambremont 1970. Caste determination in honey bees. II. Food consumption of individual honey bee larvae, determined with 32P-labeled royal jelly. Ann. Entomol. Soc. 63: 1342-1345.
Evans, J.D. and D.E. Wheeler 1999. Differential gene expression between developing queens and workers in the honey bee, Apis mellifera. Proc. Natl. Acad. Sci. 96: 5575-5580.
Foret, S., R. Kucharski, M. Pellegrini, S. Feng, S.E. Jacobsen, G.E. Robinson and R. Maleszka 2012. DNA methylation dynamics, metabolic fluxes, gene splicing and alternative phenotypes in honey bees. Proc. Natl. Acad. Sci. USA 109: 4968-4973.
Guo, X., S. Su, G. Skogerboe, S. Dai, W. Li, Z. Li, and F. Liu, 2013. Recipe for a busy bee: microRNAs in honey bee caste determination. PLoS ONE 8(12): e81661 https//doi.org110.1371/journal.pone.0081661
Mao, W., M.A. Schuler and M.R. Berenbaum 2015. A dietary phytochemical alters caste-associated gene expression in honey bees. Sci. Adv. 1:e1500795
Mutti, N.S., A.G. Dolezal, F. Wolschin, J.S. Mutti, K.S. Gill, and G.V. Amdam 2011. IRS and TOR nutrient-signaling pathways act via juvenile hormone to influence honey bee caste fate. J. Exp. Biol. 214: 3977-3984.
Rachinsky, A. and K. Hartfelder 1990. Corpora allata activity, a prime regulating element for caste-specific juvenile hormone titre in honey bee larvae (Apis mellifera carnica). J. Insect Physiol. 36: 189-194.
Wheeler, D.E., N. Buck and J.D. Evans 2006. Expression of insulin pathway genes during the period of caste determination in the honey bee, Apis mellifera. Insect Mol. Biol. 15: 597-602.
Wheeler, D.E., N.A. Buck and J.D. Evans 2014. Expression of insulin/insulin-like signaling and TOR pathway genes in honey bee caste determination. Insect Mol. Biol. 23: 113-121.
Wojciechowski, M., R. Lowe, J. Maleszka, D. Conn, R. Maleszka, and P.J. Hurd 2018. Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development. Genome Res. 28: 1532-1542.
Clarence Collison is an Emeritus Professor of Entomology and Department Head Emeritus of Entomology and Plant Pathology at Mississippi State University, Mississippi State, MS.
