Recipe for a Busy Bee: MicroRNAs in Honey Bee Caste Determination

Social 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. We have employed Illumina/Solexa sequencing to examine the small RNA content in the bee larval food, and show that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were 7–215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the 4^th to 6^th day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee.     

Mitochondrial structure and dynamics as critical factors in honey bee (Apis mellifera L.) caste development

The relationship between nutrition and phenotype is an especially challenging question in cases of facultative polyphenism, like the castes of social insects. In the honey bee, Apis mellifera, unexpected modifications in conserved signaling pathways revealed the hypoxia response as a possible mechanism underlying the regulation of body size and organ growth. Hence, the current study was designed to investigate possible causes of why the three hypoxia core genes are overexpressed in worker larvae. Parting from the hypothesis that this has an endogenous cause and is not due to differences in external oxygen levels we investigated mitochondrial numbers and distribution, as well as mitochondrial oxygen consumption rates in fat body cells of queen and worker larvae during the caste fate-critical larval stages. By immunofluorescence and electron microscopy we found higher densities of mitochondria in queen larval fat body, a finding further confirmed by a citrate synthase assay quantifying mitochondrial functional units. Oxygen consumption measurements by high-resolution respirometry revealed that queen larvae have higher maximum capacities of ATP production at lower physiological demand. Finally, the expression analysis of mitogenesis-related factors showed that the honey bee TFB1 and TFB2 homologs, and a nutritional regulator, ERR, are overexpressed in queen larvae. These results are strong evidence that the differential nutrition of queen and worker larvae by nurse bees affects mitochondrial dynamics and functionality in the fat body of these larvae, hence explaining their differential hypoxia response.     

Behavior and molecular physiology of nurses of worker and queen larvae in honey bees (Apis mellifera)

In a honey bee colony, worker bees rear a new queen by providing her with a larger cell in which to develop and a large amount of richer food (royal jelly). Royal jelly and worker jelly (fed to developing worker larvae) differ in terms of sugar, vitamin, protein and nucleotide composition. Here we examined whether workers attending queen and worker larvae are separate specialized sub-castes of the nurse bees. We collected nurse bees attending queen larvae (AQL) and worker larvae (AWL) and compared gene expression profiles of hypopharyngeal gland tissues, using Solexa/Illumina digital gene expression tag profiling (DGE). Significant differences in gene expression were found that included a disproportionate number of genes involved in glandular secretion and royal jelly synthesis. However behavioral observations showed that these were not two entirely distinct populations. Nurse workers were observed attending both worker larvae and queen larvae, and there was no evidence of a specialized group of workers that preferentially or exclusively attended developing queens. Nevertheless, AQL attended larvae more frequently compared to AWL, suggesting that nurses sampled attending queen larvae may have been the most active nurses. This study serves as another example of the relationship between differences in gene expression and behavioral specialisation in honey bees.     

Larval and nurse worker control of developmental plasticity and the evolution of honey bee queen-worker dimorphism

Social evolution in honey bees has produced strong queen-worker dimorphism for plastic traits that depend on larval nutrition. The honey bee developmental programme includes both larval components that determine plastic growth responses to larval nutrition and nurse components that regulate larval nutrition. We studied how these two components contribute to variation in worker and queen body size and ovary size for two pairs of honey bee lineages that show similar differences in worker body-ovary size allometry but have diverged over different evolutionary timescales. Our results indicate that the lineages have diverged for both nurse and larval developmental components, that rapid changes in worker body-ovary size allometry may disrupt queen development and that queen-worker dimorphism arises mainly from discrete nurse-provided nutritional environments, not from a developmental switch that converts variable nutritional environments into discrete phenotypes. Both larval and nurse components have likely contributed to the evolution of queen-worker dimorphism.     

Behavioural,physiological and molecular changes in alloparental caregivers may be responsible for selection response for female reproductive investment in honey bees

Reproductive investment is a central life history variable that influences all aspects of life. Hormones coordinate reproduction in multicellular organisms, but the mechanisms controlling the collective reproductive investment of social insects are largely unexplored. One important aspect of honey bee (Apis mellifera) reproductive investment consists of raising female‐destined larvae into new queens by alloparental care of nurse bees in form of royal jelly provisioning. Artificial selection for commercial royal jelly production over 40 years has increased this reproductive investment by an order of magnitude. In a cross‐fostering experiment, we establish that this shift in social phenotype is caused by nurse bees. We find no evidence for changes in larval signalling. Instead, the antennae of the nurse bees of the selected stock are more responsive to brood pheromones than control bees. Correspondingly, the selected royal jelly bee nurses are more attracted to brood pheromones than unselected control nurses. Comparative proteomics of the antennae from the selected and unselected stocks indicate putative molecular mechanisms, primarily changes in chemosensation and energy metabolism. We report expression differences of several candidate genes that correlate with the differences in reproductive investment. The functional relevance of these genes is supported by demonstrating that the corresponding proteins can competitively bind one previously described and one newly discovered brood pheromone. Thus, we suggest several chemosensory genes, most prominently OBP16 and CSP4, as candidate mechanisms controlling queen rearing, a key reproductive investment, in honey bees. These findings reveal novel aspects of pheromonal communication in honey bees and explain how sensory changes affect communication and lead to a drastic shift in colony‐level resource allocation to sexual reproduction. Thus, pheromonal and hormonal communication may play similar roles for reproductive investment in superorganisms and multicellular organisms, respectively.     

A comparison of honeybee (Apis mellifera) queen,worker and drone larvae by RNA‐Seq

Honeybees (Apis mellifera) have haplodiploid sex determination: males develop from unfertilized eggs and females develop from fertilized ones. The differences in larval food also determine the development of females. Here we compared the total somatic gene expression profiles of 2-day and 4-day-old drone, queen and worker larvae by RNASeq. The results from a co-expression network analysis on all expressed genes showed that 2-day-old drone and worker larvae were closer in gene expression profiles than 2-day-old queen larvae. This indicated that for young larvae (2-day-old) environmental factors such as larval diet have a greater effect on gene expression profiles than ploidy or sex determination. Drones had the most distinct gene expression profiles at the 4-day larval stage, suggesting that haploidy, or sex dramatically affects the gene expression of honeybee larvae. Drone larvae showed fewer differences in gene expression profiles at the 2-day and 4-day time points than the worker and queen larval comparisons (598 against 1190 and 1181), suggesting a different pattern of gene expression regulation during the larval development of haploid males compared to diploid females. This study indicates that early in development the queen caste has the most distinct gene expression profile, perhaps reflecting the very rapid growth and morphological specialization of this caste compared to workers and drones. Later in development the haploid male drones have the most distinct gene expression profile, perhaps reflecting the influence of ploidy or sex determination on gene expression.     

The effects of honey bee (Apis mellifera L.) queen reproductive potential on colony growth

Reproduction in species of eusocial insects is monopolized by one or a few individuals, while the remaining colony tasks are performed by the worker caste. This reproductive division of labor is exemplified by honey bees (Apis mellifera L.), in which a single, polyandrous queen is the sole colony member that lays fertilized eggs. Previous work has revealed that the developmental fate of honey bee queens is highly plastic, with queens raised from younger worker larvae exhibiting higher measures in several aspects of reproductive potential compared to queens raised from older worker larvae. Here, we investigated the effects of queen reproductive potential (“quality”) on the growth and winter survival of newly established honey bee colonies. We did so by comparing the growth of colonies headed by “high-quality” queens (i.e., those raised from young worker larvae, which are more queen-like morphologically) to those headed by “low-quality” queens (i.e., those raised from older worker larvae, which are more worker-like morphologically). We confirmed that queens reared from young worker larvae were significantly larger in size than queens reared from old worker larvae. We also found a significant positive effect of queen grafting age on a colony’s production of worker comb, drone comb, and stored food (honey and pollen), although we did not find a statistically significant difference in the production of worker and drone brood, worker population, and colony weight. Our results provide evidence that in honey bees, queen developmental plasticity influences several important measures of colony fitness. Thus, the present study supports the idea that a honey bee colony can be viewed (at least in part) as the expanded phenotype of its queen, and thus selection acting predominantly at the colony level can be congruent with that at the individual level.     

Larval juvenile hormone treatment affects pre-adult development,but not adult age at onset of foraging in worker honey bees (Apis mellifera)

Previous research has shown that juvenile hormone (JH) titers increase as adult worker honey bees age and treatments with JH, JH analogs and JH mimics induce precocious foraging. Larvae from genotypes exhibiting faster adult behavioral development had significantly higher levels of juvenile hormone during the 2nd and 3rd larval instar. It is known that highly increased JH during this period causes the totipotent female larvae to differentiate into a queen. We treated third instar larvae with JH to test the hypothesis that this time period may be a developmental critical period for organizational effects of JH on brain and behavior also in the worker caste, such that JH treatment at a lower level than required to produce queens will speed adult behavioral development in workers. Larval JH treatment did not influence adult worker behavioral development. However, it made pre-adult development more queen-like in two ways: treated larvae were capped sooner by adult bees, and emerged from pupation earlier. These results suggest that some aspects of honey bee behavioral development may be relatively insensitive to pre-adult perturbation. These results also suggest JH titer may be connected to cues perceived by the adult bees indicating larval readiness for pupation resulting in adult bee cell capping behavior.     

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies.     

Making a queen: an epigenetic analysis of the robustness of the honeybee (Apis mellifera) queen developmental pathway

Specialized castes are considered a key reason for the evolutionary and ecological success of the social insect lifestyle. The most essential caste distinction is between the fertile queen and the sterile workers. Honeybee (Apis mellifera) workers and queens are not genetically distinct, rather these different phenotypes are the result of epigenetically regulated divergent developmental pathways. This is an important phenomenon in understanding the evolution of social insect societies. Here, we studied the genomic regulation of the worker and queen developmental pathways, and the robustness of the pathways by transplanting eggs or young larvae to queen cells. Queens could be successfully reared from worker larvae transplanted up to 3 days age, but queens reared from older worker larvae had decreased queen body size and weight compared with queens from transplanted eggs. Gene expression analysis showed that queens raised from worker larvae differed from queens raised from eggs in the expression of genes involved in the immune system, caste differentiation, body development and longevity. DNA methylation levels were also higher in 3‐day‐old queen larvae raised from worker larvae compared with that raised from transplanted eggs identifying a possible mechanism stabilizing the two developmental paths. We propose that environmental (nutrition and space) changes induced by the commercial rearing practice result in a suboptimal queen phenotype via epigenetic processes, which may potentially contribute to the evolution of queen–worker dimorphism. This also has potentially contributed to the global increase in honeybee colony failure rates.     

The role of exogenous JH I,JH III and Anti-JH (precocene II) on queen induction of 4.5-day-old worker honey bee larvae

Worker larvae at an age of 4½ days were fed one of several mixtures of reconstituted royal jelly adjusted to a refractive index of 1.3825 and supplemented with JH I, JH III or Anti-JH (precocene II). In addition, juvenile hormone was topically applied to larvae of the same age. It was readily apparent that caste induction is concentration-dependent and that 4?-day-old worker larvae can still develop into queens under laboratory conditions, providing that they have not stopped feeding or can be induced to commence feeding again. These findings are contrary to the general belief that queen induction is not possible after a socalled sensitive period of 3–3½ days. Queens resulted only from honey bee larvae exposed to royal jelly containing 1 μg of JH I. In addition, oral application at this concentration resulted in the only case in which the normal mean weights of worker honey bees were exceeded. All other concentrations of juvenile hormone were not sufficient to initiate queen induction, although its lower concentration may have influenced the production of intercastes.Precocene II did not play a role in queen induction and it also did not interfere with the growth of developing larvae or adults. In addition, the lack of malformations in honey bees treated with precocene II indicates that the use of such a compound as a control agent in insect populations will probably not be detrimental to honey bee larvae that are at least 4½ days old. However, large doses of precocene will quickly kill most 3½-day-old honey bee larvae.The evidence presented here clearly indicates that caste determination is regulated by the endocrine system in honey bee larvae. Food intake in honey bee larvae may well be regulated by the endocrine system. Thus, an apparently inhibited corpus allatum (C.A.) could be reactivated by food intake coupled with juvenile hormone. The food intake restriction that worker larvae normally encounter in the hive probably results in a cessation of C.A. activity. The increase in food intake by queen larvae, on the other hand, carries an increase in growth and accompanying morphological changes necessary for queen development. This concept may also explain the development of intercastes encountered in in vitro studies. Only those larvae that follow a normal food intake sequence, i.e. moderate during the first 3–4 days or so, will develop into queens. Conversely, those larvae that take in too much food during the early portion of development may achieve incomplete development of the neurosecretory system and, thus, develop into intercastes.     

蜜蜂级型分化机理

蜜蜂Apis spp.能有效地为多种植物及农作物授粉, 具有重要的经济和生态价值; 蜜蜂作为高度真社会性昆虫, 已成为社会生物学研究的模式生物。社会性昆虫的生殖劳动分工具有重要的进化意义, 而级型分化是形成生殖劳动分工的基础。近年来, 关于蜜蜂级型分化的研究已取得诸多重要成果, 其机理也得到了较为深入的阐释。营养差异引发蜜蜂幼虫的级型分化。蜂王浆中的主要蛋白组分之一--Royalactin是诱导蜂王发育的关键营养因子, 而脂肪体细胞的表皮生长因子受体介导了Royalactin的这种蜂王诱导作用。DNA甲基化是重要的表观遗传机制之一, 且与个体发育和疾病发生紧密相关, 近来的研究表明DNA甲基化在蜜蜂级型分化过程中发挥重要的调控作用。此外, 越来越多的研究进一步深化了人们对内分泌系统调节级型分化作用的认识。本文从关键营养因子调控、 表观遗传调控和内分泌调节3方面综述蜜蜂级型分化的机理, 并对未来的研究提出可能的方向。     

Caste Determination in Bombus terrestris: Differences in Development and Rates of JH Biosynthesis between Queen and Worker Larvae

To study the possible role of juvenile hormone in caste determination in Bombus terrestris, we measured development and rates of juvenile hormone biosynthesis in vitro in larvae destined to develop into either workers or queens. Larvae of both castes developed through four instars and had the same growth rates. However, the duration of the instars was longer for queen larvae, and their head width at the third and fourth instars was significantly larger. After validating the well-known radiochemical assay of JH for bumble bee larvae, we show that worker larvae corpora allata exhibited a constant and low rate of JH biosynthesis, never more than 5 pmol JH/h/pair. Queen larvae, in contrast, had two peaks of JH biosynthesis: a small one during the first instar, which has previously been correlated with caste determination; and a large peak, previously undetected, above 40 pmol JH/h/pair, during the second and third instars. We suggest that caste determination in this species is mediated by JH and that the duration of larval instars is a key factor. The possibility that the queen influences caste determination via an effect on instar duration is also discussed. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Diet and cell size both affect queen-worker differentiation through DNA methylation in honey bees (Apis mellifera, Apidae)

Background

Young larvae of the honey bee (Apis mellifera) are totipotent; they can become either queens (reproductives) or workers (largely sterile helpers). DNA methylation has been shown to play an important role in this differentiation. In this study, we examine the contributions of diet and cell size to caste differentiation.

Methodology/Principal Findings

We measured the activity and gene expression of one key enzyme involved in methylation, Dnmt3; the rates of methylation in the gene dynactin p62; as well as morphological characteristics of adult bees developed either from larvae fed with worker jelly or royal jelly; and larvae raised in either queen or worker cells. We show that both diet type and cell size contributed to the queen-worker differentiation, and that the two factors affected different methylation sites inside the same gene dynactin p62.

Conclusions/Significance

We confirm previous findings that Dnmt3 plays a critical role in honey bee caste differentiation. Further, we show for the first time that cell size also plays a role in influencing larval development when diet is kept the same.     

Honey bee PTEN--description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes

Background

Phosphatase and TENsin (PTEN) homolog is a negative regulator that takes part in IIS (insulin/insulin-like signaling) and Egfr (epidermal growth factor receptor) activation in Drosophila melanogaster. IIS and Egfr signaling events are also involved in the developmental process of queen and worker differentiation in honey bees (Apis mellifera). Here, we characterized the bee PTEN gene homologue for the first time and begin to explore its potential function during bee development and adult life.

Results

Honey bee PTEN is alternatively spliced, resulting in three splice variants. Next, we show that the expression of PTEN can be down-regulated by RNA interference (RNAi) in the larval stage, when female caste fate is determined. Relative to controls, we observed that RNAi efficacy is dependent on the amount of PTEN dsRNA that is delivered to larvae. For larvae fed queen or worker diets containing a high amount of PTEN dsRNA, PTEN knockdown was significant at a whole-body level but lethal. A lower dosage did not result in a significant gene down-regulation. Finally, we compared same-aged adult workers with different behavior: nursing vs. foraging. We show that between nurses and foragers, PTEN isoforms were differentially expressed within brain, ovary and fat body tissues. All isoforms were expressed at higher levels in the brain and ovaries of the foragers. In fat body, isoform B was expressed at higher level in the nurse bees.

Conclusion

Our results suggest that PTEN plays a central role during growth and development in queen- and worker-destined honey bees. In adult workers, moreover, tissue-specific patterns of PTEN isoform expression are correlated with differences in complex division of labor between same-aged individuals. Therefore, we propose that knowledge on the roles of IIS and Egfr activity in developmental and behavioral control may increase through studies of how PTEN functions can impact bee social phenotypes.     

Development and evolution of caste dimorphism in honeybees – a modeling approach

The difference in phenotypes of queens and workers is a hallmark of the highly eusocial insects. The caste dimorphism is often described as a switch‐controlled polyphenism, in which environmental conditions decide an individual's caste. Using theoretical modeling and empirical data from honeybees, we show that there is no discrete larval developmental switch. Instead, a combination of larval developmental plasticity and nurse worker feeding behavior make up a colony‐level social and physiological system that regulates development and produces the caste dimorphism. Discrete queen and worker phenotypes are the result of discrete feeding regimes imposed by nurses, whereas a range of experimental feeding regimes produces a continuous range of phenotypes. Worker ovariole numbers are reduced through feeding‐regime‐mediated reduction in juvenile hormone titers, involving reduced sugar in the larval food. Based on the mechanisms identified in our analysis, we propose a scenario of the evolutionary history of honeybee development and feeding regimes.