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.     

Juvenile hormone in adult eusocial hymenoptera: Gonadotropin and behavioral pacemaker

Studies on the role of juvenile hormone (JH) in adult social Hymenoptera have focused on the regulation of two fundamental aspects of colony organization: reproductive division of labor between queens and workers and age-related division of labor among workers. JH acts as a gonadotropin in the primitively eusocial wasp and bumble bee species studied, and may also play this role in the advanced eusocial fire ants. However, there is no evidence that JH acts as a traditional gonadotropin in the advanced eusocial honey bee or in the few other ant species that have recently begun to be studied. The role of JH in age-related division of labor has been most thoroughly examined in honey bees. Results of these studies demonstrate that JH acts as a “behavioral pacemaker,” influencing how fast a worker grows up and makes the transition from nest activities to foraging. Hypotheses concerning the evolutionary relationship between the two functions of JH in adult eusocial Hymenoptera are discussed. Arch. Insect Biochem. Physiol. 35:559–583, 1997. © 1997 Wiley-Liss, Inc.     

Sensory response system of social behavior tied to female reproductive traits

Background

Honey bees display a complex set of anatomical, physiological, and behavioral traits that correlate with the colony storage of surplus pollen (pollen hoarding). We hypothesize that the association of these traits is a result of pleiotropy in a gene signaling network that was co-opted by natural selection to function in worker division of labor and foraging specialization. By acting on the gene network, selection can change a suite of traits, including stimulus/response relationships that affect individual foraging behavior and alter the colony level trait of pollen hoarding. The ‘pollen-hoarding syndrome’ of honey bees is the best documented syndrome of insect social organization. It can be exemplified as a link between reproductive anatomy (ovary size), physiology (yolk protein level), and foraging behavior in honey bee strains selected for pollen hoarding, a colony level trait. The syndrome gave rise to the forager-Reproductive Ground Plan Hypothesis (RGPH), which proposes that the regulatory control of foraging onset and foraging preference toward nectar or pollen was derived from a reproductive signaling network. This view was recently challenged. To resolve the controversy, we tested the associations between reproductive anatomy, physiology, and stimulus/response relationships of behavior in wild-type honey bees.

Methodology/Principal Findings

Central to the stimulus/response relationships of honey bee foraging behavior and pollen hoarding is the behavioral trait of sensory sensitivity to sucrose (an important sugar in nectar). To test the linkage of reproductive traits and sensory response systems of social behavior, we measured sucrose responsiveness with the proboscis extension response (PER) assay and quantified ovary size and vitellogenin (yolk precursor) gene expression in 6–7-day-old bees by counting ovarioles (ovary filaments) and by using semiquantitative real time RT-PCR. We show that bees with larger ovaries (more ovarioles) are characterized by higher levels of vitellogenin mRNA expression and are more responsive to sucrose solutions, a trait that is central to division of labor and foraging specialization.

Conclusions/Significance

Our results establish that in wild-type honey bees, ovary size and vitellogenin mRNA level covary with the sucrose sensory response system, an important component of foraging behavior. This finding validates links between reproductive physiology and behavioral-trait associations of the pollen-hoarding syndrome of honey bees, and supports the forager-RGPH. Our data address a current evolutionary debate, and represent the first direct demonstration of the links between reproductive anatomy, physiology, and behavioral response systems that are central to the control of complex social behavior in insects.     

Methoprene does not affect juvenile hormone titers in honey bee (Apis mellifera) workers

Methoprene, a juvenile hormone (JH) analog, is a widely used insecticide that also accelerates behavioral development in honey bees (Apis mellifera). JH regulates the transition from nursing to foraging in adult worker bees, and treatment with JH or methoprene have both been shown to induce precocious foraging. To determine how methoprene changes honey bee behavior, we compared JH titers of methoprene‐treated and untreated bees. Behavioral observations confirmed that methoprene treatment significantly increased the number of precocious foragers in 3 out of 4 colonies. In only 1 out of 4 colonies, however, was there a significant difference in JH titers between the methoprene‐treated and control bees. Further, in all 4 colonies, there was no significant differences in JH titers between precocious and normal‐aged foragers. These results suggest that methoprene did not directly affect the endogenous JH secreted by corpora allata. Because methoprene caused early foraging without changing workers’ JH titers, we conclude that methoprene most likely acts directly on the JH receptors as a substitute for JH.     

Limits on volume changes in the mushroom bodies of the honey bee brain

The behavioral maturation of adult worker honey bees is influenced by a rising titer of juvenile hormone (JH), and is temporally correlated with an increase in the volume of the neuropil of the mushroom bodies, a brain region involved in learning and memory. We explored the stability of this neuropil expansion and its possible dependence on JH. We studied the volume of the mushroom bodies in adult bees deprived of JH by surgical removal of the source glands, the corpora allata. We also asked if the neuropil expansion detected in foragers persists when bees no longer engage in foraging, either because of the onset of winter or because colony social structure was experimentally manipulated to cause some bees to revert from foraging to tending brood (nursing). Results show that adult exposure to JH is not necessary for growth of the mushroom body neuropil, and that the volume of the mushroom body neuropil in adult bees is not reduced if foraging stops. These results are interpreted in the context of a qualitative model that posits that mushroom body neuropil volume enlargement in the honey bee has both experience-independent and experience-dependent components.     

Support for the reproductive ground plan hypothesis of social evolution and major QTL for ovary traits of Africanized worker honey bees (Apis mellifera L.)

Background  

The reproductive ground plan hypothesis of social evolution suggests that reproductive controls of a solitary ancestor have been co-opted during social evolution, facilitating the division of labor among social insect workers. Despite substantial empirical support, the generality of this hypothesis is not universally accepted. Thus, we investigated the prediction of particular genes with pleiotropic effects on ovarian traits and social behavior in worker honey bees as a stringent test of the reproductive ground plan hypothesis. We complemented these tests with a comprehensive genome scan for additional quantitative trait loci (QTL) to gain a better understanding of the genetic architecture of the ovary size of honey bee workers, a morphological trait that is significant for understanding social insect caste evolution and general insect biology.     

Juvenile hormone paces behavioral development in the adult worker honey bee

Behavioral development in the adult worker honey bee (Apis mellifera), from performing tasks inside the hive to foraging, is associated with an increase in the blood titer of juvenile hormone III (JH), and hormone treatment results in precocious foraging. To study behavioral development in the absence of JH we removed its glandular source, the corpora allata, in 1-day-old adult bees. The age at onset of foraging for allatectomized bees in typical colonies was significantly older compared with that of sham-operated bees in 3 out of 4 colonies; this delay was eliminated by hormone replacement in 3 out of 3 colonies. To determine the effects of corpora allata removal on sensitivity to changes in conditions that influence the rate of behavioral development, we used "single-cohort" colonies (composed of only young bees) in which some colony members initiate foraging precociously. The age at onset of foraging for allatectomized bees was significantly older compared with that of sham-operated bees in 2 out of 3 colonies, and this delay was eliminated by hormone replacement. Allatectomized bees initiated foraging at significantly younger ages in single-cohort colonies than in typical colonies. These results demonstrate that JH influences the pace of behavioral development in honey bees, but is not essential for either foraging or altering behavioral development in response to changes in conditions.     

Juvenile hormone and circadian locomotor activity in the honey bee Apis mellifera

Age-related division of labor in honeybees is associated with plasticity in circadian rhythms. Young nest bees care for brood around the clock with no circadian rhythms while older foragers have strong circadian rhythms that are used for sun compass navigation and for timing visits to flowers. Since juvenile hormone (JH) is involved in the coordination of physiological and behavioral processes underlying age-related division of labor in honey bees, we tested the hypothesis that JH influences the ontogeny of circadian rhythms and other clock parameters in young worker bees. Treatments with the JH analog methoprene or allatectomy did not influence the onset of rhythmicity, overall locomotor activity, or the free-running period of rhythmic locomotor behavior. There were, however, significant differences in the onset of rhythmicity, overall locomotor activity, and longevity between bees from different source colonies, suggesting that there is significant genetic variation for these traits. Our results suggest that JH does not coordinate all aspects of division of labor in bees and that coordination of task performance with circadian rhythms is probably mediated by other regulatory systems.     

Experience-dependent plasticity in the mushroom bodies of the solitary bee Osmia lignaria (Megachilidae)

All members of the solitary bee species Osmia lignaria (the orchard bee) forage upon emergence from their natal nest cell. Conversely, in the honey bee, days-to-weeks of socially regulated behavioral development precede the onset of foraging. The social honey bee's behavioral transition to foraging is accompanied by neuroanatomical changes in the mushroom bodies, a region of the insect brain implicated in learning. If these changes were general adaptations to foraging, they should also occur in the solitary orchard bee. Using unbiased stereological methods, we estimated the volume of the major compartments of the mushroom bodies, the neuropil and Kenyon cell body region, in adult orchard bees. We compared the mushroom bodies of recently emerged bees with mature bees that had extensive foraging experience. To separate effects of general maturation from field foraging, some orchard bees were confined to a cage indoors. The mushroom body neuropil of experienced field foragers was significantly greater than that of both recently emerged and mature caged orchard bees, suggesting that, like the honey bee, this increase is driven by outdoor foraging experience. Unlike the honey bee, where increases in the ratio of neuropil to Kenyon cell region occur in the worker after emerging from the hive cell, the orchard bee emerged from the natal nest cell with a ratio that did not change with maturation and was comparable to honey-bee foragers. These results suggest that a common developmental endpoint may be reached via different development paths in social and solitary species of foraging bees.     

Conservation in Mammals of Genes Associated with Aggression-Related Behavioral Phenotypes in Honey Bees

The emerging field of sociogenomics explores the relations between social behavior and genome structure and function. An important question is the extent to which associations between social behavior and gene expression are conserved among the Metazoa. Prior experimental work in an invertebrate model of social behavior, the honey bee, revealed distinct brain gene expression patterns in African and European honey bees, and within European honey bees with different behavioral phenotypes. The present work is a computational study of these previous findings in which we analyze, by orthology determination, the extent to which genes that are socially regulated in honey bees are conserved across the Metazoa. We found that the differentially expressed gene sets associated with alarm pheromone response, the difference between old and young bees, and the colony influence on soldier bees, are enriched in widely conserved genes, indicating that these differences have genomic bases shared with many other metazoans. By contrast, the sets of differentially expressed genes associated with the differences between African and European forager and guard bees are depleted in widely conserved genes, indicating that the genomic basis for this social behavior is relatively specific to honey bees. For the alarm pheromone response gene set, we found a particularly high degree of conservation with mammals, even though the alarm pheromone itself is bee-specific. Gene Ontology identification of human orthologs to the strongly conserved honey bee genes associated with the alarm pheromone response shows overrepresentation of protein metabolism, regulation of protein complex formation, and protein folding, perhaps associated with remodeling of critical neural circuits in response to alarm pheromone. We hypothesize that such remodeling may be an adaptation of social animals to process and respond appropriately to the complex patterns of conspecific communication essential for social organization.     

Effects of selection for honey bee worker reproduction on foraging traits

The “reproductive ground plan” hypothesis (RGPH) proposes that reproductive division of labour in social insects had its antecedents in the ancient gene regulatory networks that evolved to regulate the foraging and reproductive phases of their solitary ancestors. Thus, queens express traits that are characteristic of the reproductive phase of solitary insects, whereas workers express traits characteristic of the foraging phase. The RGPH has also been extended to help understand the regulation of age polyethism within the worker caste and more recently to explain differences in the foraging specialisations of individual honey bee workers. Foragers that specialise in collecting proteinaceous pollen are hypothesised to have higher reproductive potential than individuals that preferentially forage for nectar because genes that were ancestrally associated with the reproductive phase are active. We investigated the links between honey bee worker foraging behaviour and reproductive traits by comparing the foraging preferences of a line of workers that has been selected for high rates of worker reproduction with the preferences of wild-type bees. We show that while selection for reproductive behaviour in workers has not altered foraging preferences, the age at onset of foraging of our selected line has been increased. Our findings therefore support the hypothesis that age polyethism is related to the reproductive ground plan, but they cast doubt on recent suggestions that foraging preferences and reproductive traits are pleiotropically linked.     

Behavioral rhythmicity, age, division of labor and period expression in the honey bee brain.

Young adult honey bees work inside the beehive "nursing" brood around the clock with no circadian rhythms; older bees forage for nectar and pollen outside with strong circadian rhythms. Previous research has shown that the development of an endogenous rhythm of activity is also seen in the laboratory in a constant environment. Newly emerging bees maintained in isolation are typically arrhythmic during the first few days of adult life and develop strong circadian rhythms by about a few days of age. In addition, average daily levels of period (per) mRNA in the brain are higher in foragers or forager-age bees (> 21 days of age) relative to young nest bees (approximately 7 days of age). The authors used social manipulations to uncouple behavioral rhythmicity, age, and task to determine the relationship between these factors and per. There was no obligate link between average daily levels of per brain mRNA and either behavioral rhythmicity or age. There also were no differences in per brain mRNA levels between nurse bees and foragers in social environments that promote precocious or reversed behavioral development. Nurses and other hive-age bees can have high or low levels of per mRNA levels in the brain, depending on the social environment, while foragers and foraging-age bees always have high levels. These findings suggest a link between honey bee foraging behavior and per up-regulation. Results also suggest task-related differences in the amplitude of per mRNA oscillation in the brain, with foragers having larger diurnal fluctuation in per than nurses, regardless of age. Taken together, these results suggest that social factors may exert potent influences on the regulation of clock genes.     

Colony Integration in Honey Bees: Mechanisms of Behavioral Reversion

After confirming that worker honey bees (Apis mellifera) can revert from foraging to brood care, we determined whether juvenile hormone (JH) mediates this form of plasticity in behavioral development and whether worker age and genotype influence the probability of its expression. Measurements of JH titers support the hypothesis that plasticity in honey bee behavioral development is a consequence of modulation of JH by extrinsic factors. Observations of individually marked bees in a colony composed of two phenotypically distinguishable subfamilies revealed that the likelihood of undergoing behavioral reversion was influenced by worker age but not by worker genotype. The effect of worker age on reversion is consistent with a previously formulated model for the regulation of age polyethism in honey bees that predicts that workers of different ages have different response thresholds for task-associated stimuli. The lack of a genotypic effect on reversion is in contrast to results for other forms of behavioral plasticity.     

后基因组时代的蜜蜂QTL研究

继果蝇、按蚊和家蚕之后,意大利蜜蜂Apis mellifera（膜翅目： 蜜蜂科）成为又一种被完整测得基因组序列的昆虫。从此,蜜蜂研究进入后基因组时代。作为一种典型的社会性昆虫,许多和蜜蜂社会生活紧密相关的性状都是数量性状。这些性状研究中广泛涉及到了数量性状位点（quantitative traits loci,QTL）定位研究。本文综述了应用QTL对蜜蜂取食行为、自卫行为、体长、逆转学习等的研究现状,同时结合国内外最新研究进展,总结并展望了后基因组时代蜜蜂QTL的研究方向。     

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.     

Genetic variation in worker temporal polyethism and colony defensiveness in the honey bee, Apis mellifera

To test the hypothesis that colonies of honey bees composedof workers with faster rates of adult behavioral developmentare more defensive than colonies composed of workers with slowerbehavioral development, we determined whether there is a correlationbetween genetic variation in worker temporal polyethism andcolony defensiveness. There was a positive correlation for thesetwo traits, both for European and Africanized honey bees. Thecorrelation was larger for Africanized bees, due to differencesbetween Africanized and European bees, differences in experimentaldesign, or both. Consistent with these results was the findingthat colonies with a higher proportion of older bees were moredefensive than colonies of the same size that had a lower proportionof older bees. There also was a positive correlation betweenrate of individual behavioral development and the intensityof colony flight activity, and a negative correlation betweencolony defensiveness and flight activity. This suggests thatthe relationship between temporal polyethism and colony defensivenessmay vary with the manner in which foraging and defense dutiesare allocated among a colony's older workers. These resultsindicate that genotypic differences in rates of worker behavioraldevelopment can influence the phenotype of a honey bee colonyin a variety of ways.     

No Genetic Tradeoffs between Hygienic Behaviour and Individual Innate Immunity in the Honey Bee,Apis mellifera

Many animals have individual and social mechanisms for combating pathogens. Animals may exhibit short-term physiological tradeoffs between social and individual immunity because the latter is often energetically costly. Genetic tradeoffs between these two traits can also occur if mutations that enhance social immunity diminish individual immunity, or vice versa. Physiological tradeoffs between individual and social immunity have been previously documented in insects, but there has been no study of genetic tradeoffs involving these traits. There is strong evidence that some genes influence both innate immunity and behaviour in social insects – a prerequisite for genetic tradeoffs. Quantifying genetic tradeoffs is critical for understanding the evolution of immunity in social insects and for devising effective strategies for breeding disease-resistant pollinator populations. We conducted two experiments to test the hypothesis of a genetic tradeoff between social and individual immunity in the honey bee, Apis mellifera. First, we estimated the relative contribution of genetics to individual variation in innate immunity of honey bee workers, as only heritable traits can experience genetic tradeoffs. Second, we examined if worker bees with hygienic sisters have reduced individual innate immune response. We genotyped several hundred workers from two colonies and found that patriline genotype does not significantly influence the antimicrobial activity of a worker’s hemolymph. Further, we did not find a negative correlation between hygienic behaviour and the average antimicrobial activity of a worker’s hemolymph across 30 honey bee colonies. Taken together, our work indicates no genetic tradeoffs between hygienic behaviour and innate immunity in honey bees. Our work suggests that using artificial selection to increase hygienic behaviour of honey bee colonies is not expected to concurrently compromise individual innate immunity of worker bees.