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Highly social insects are characterized by caste dimorphism, with distinct size differences of reproductive organs between fertile queens and the more or less sterile workers. An abundance of nutrition or instruction via diet-specific compounds has been proposed as explanations for the nutrition-driven queen and worker polyphenism. Here, we further explored these models in the honeybee (Apis mellifera) using worker nutrition rearing and a novel mutational screening approach using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. The worker nutrition-driven size reduction of reproductive organs was restricted to the female sex, suggesting input from the sex determination pathway. Genetic screens on the sex determination genes in genetic females for size polyphenism revealed that doublesex (dsx) mutants display size-reduced reproductive organs irrespective of the sexual morphology of the organ tissue. In contrast, feminizer (fem) mutants lost the response to worker nutrition-driven size control. The first morphological worker mutants in honeybees demonstrate that the response to nutrition relies on a genetic program that is switched \"ON\" by the fem gene. Thus, the genetic instruction provided by the fem gene provides an entry point to genetically dissect the underlying processes that implement the size polyphenism.

Honey bees (Apis mellifera) provide a principal example of diphenic development. Excess feeding of female larvae results in queens (large reproductives). Moderate diet yields workers (small helpers). The signaling pathway that links provisioning to female developmental fate is not understood, yet we reasoned that it could include TOR (target of rapamycin), a nutrient- and energy-sensing kinase that controls organismal growth. Here, the role of Apis mellifera TOR (amTOR) in caste determination is examined by rapamycin/FK506 pharmacology and RNA interference (RNAi) gene knockdown. We show that in queen-destined larvae, the TOR inhibitor rapamycin induces the development of worker characters that are blocked by the antagonist FK506. Further, queen fate is associated with elevated activity of the Apis mellifera TOR encoding gene, amTOR, and amTOR gene knockdown blocks queen fate and results in individuals with worker morphology. A much-studied insect dimorphism, thereby, can be governed by the TOR pathway. Our results present the first evidence for a role of TOR in diphenic development, and suggest that adoption of this ancestral nutrient-sensing cascade is one evolutionary pathway for morphological caste differentiation in social insects.

Food choice and eating behavior affect health and longevity. Large-scale research efforts aim to understand the molecular and social/behavioral mechanisms of energy homeostasis, body weight, and food intake. Honey bees (Apis mellifera) could provide a model for these studies since individuals vary in food-related behavior and social factors can be controlled. Here, we examine a potential role of peripheral insulin receptor substrate (IRS) expression in honey bee foraging behavior. IRS is central to cellular nutrient sensing through transduction of insulin/insulin-like signals (IIS). By reducing peripheral IRS gene expression and IRS protein amount with the use of RNA interference (RNAi), we demonstrate that IRS influences foraging choice in two standard strains selected for different food-hoarding behavior. Compared with controls, IRS knockdowns bias their foraging effort toward protein (pollen) rather than toward carbohydrate (nectar) sources. Through control experiments, we establish that IRS does not influence the bees' sucrose sensory response, a modality that is generally associated with food-related behavior and specifically correlated with the foraging preference of honey bees. These results reveal a new affector pathway of honey bee social foraging, and suggest that IRS expressed in peripheral tissue can modulate an insect's foraging choice between protein and carbohydrate sources.

In vitro rearing is an important and useful tool for honey bee (Apis mellifera L.) studies. However, it often results in intercastes between queens and workers, which are normally are not seen in hive-reared bees, except when larvae older than three days are grafted for queen rearing. Morphological classification (queen versus worker or intercastes) of bees produced by this method can be subjective and generally depends on size differences. Here, we propose an alternative method for caste classification of female honey bees reared in vitro, based on weight at emergence, ovariole number, spermatheca size and size and shape, and features of the head, mandible and basitarsus. Morphological measurements were made with both traditional morphometric and geometric morphometrics techniques. The classifications were performed by principal component analysis, using naturally developed queens and workers as controls. First, the analysis included all the characters. Subsequently, a new analysis was made without the information about ovariole number and spermatheca size. Geometric morphometrics was less dependent on ovariole number and spermatheca information for caste and intercaste identification. This is useful, since acquiring information concerning these reproductive structures requires time-consuming dissection and they are not accessible when abdomens have been removed for molecular assays or in dried specimens. Additionally, geometric morphometrics divided intercastes into more discrete phenotype subsets. We conclude that morphometric geometrics are superior to traditional morphometrics techniques for identification and classification of honey bee castes and intermediates.

In animals, dietary restriction or suppression of genes involved in nutrient sensing tends to increase lifespan. In contrast, food restriction in honeybees (Apis mellifera) shortens lifespan by accelerating a behavioural maturation program that culminates in leaving the nest as a forager. Foraging is metabolically demanding and risky, and foragers experience increased rates of aging and mortality. Food-deprived worker bees forage at younger ages and are expected to live shorter lives. We tested whether suppression of a molecular nutrient sensing pathway is sufficient to accelerate the behavioural transition to foraging and shorten worker life. To achieve this, we reduced expression of the insulin receptor substrate (irs) gene via RNA interference in two selected lines of honeybees used to control for behavioural and genetic variation. irs encodes a membrane-associated protein in the insulin/insulin-like signalling (IIS) pathway that is central to nutrient sensing in animals. We measured foraging onset and lifespan and found that suppression of irs reduced worker bee lifespan in both genotypes, and that this effect was largely driven by an earlier onset of foraging behaviour in a genotype-conditional manner. Our results provide the first direct evidence that an IIS pathway gene influences behavioural maturation and lifespan in honeybees and highlight the importance of considering social environments and behaviours when investigating the regulation of aging and lifespan in social animals.

Ovarioles are the functional unit of the female insect reproductive organs and the number of ovarioles per ovary strongly influences egg-laying rate and fecundity. Social evolution in the honeybee (Apis mellifera) has resulted in queens with 200–360 total ovarioles and workers with usually 20 or less. In addition, variation in ovariole number among workers relates to worker sensory tuning, foraging behavior, and the ability to lay unfertilized male-destined eggs. To study the genetic architecture of worker ovariole number, we performed a series of crosses between Africanized and European bees that differ in worker ovariole number. Unexpectedly, these crosses produced transgressive worker phenotypes with extreme ovariole numbers that were sensitive to the social environment. We used a new selective pooled DNA interval mapping approach with two Africanized backcrosses to identify quantitative trait loci (QTL) underlying the transgressive ovary phenotype. We identified one QTL on chromosome 11 and found some evidence for another QTL on chromosome 2. Both QTL regions contain plausible functional candidate genes. The ovariole number of foragers was correlated with the sugar concentration of collected nectar, supporting previous studies showing a link between worker physiology and foraging behavior. We discuss how the phenotype of extreme worker ovariole numbers and the underlying genetic factors we identified could be linked to the development of queen traits.

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. 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. 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.

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. Results Back-crossing hybrid European x Africanized honey bee queens to the Africanized parent colony generated two study populations with extraordinarily large worker ovaries. Despite the transgressive ovary phenotypes, several previously mapped QTL for social foraging behavior demonstrated ovary size effects, confirming the prediction of pleiotropic genetic effects on reproductive traits and social behavior. One major QTL for ovary size was detected in each backcross, along with several smaller effects and two QTL for ovary asymmetry. One of the main ovary size QTL coincided with a major QTL for ovary activation, explaining 3/4 of the phenotypic variance, although no simple positive correlation between ovary size and activation was observed. Conclusions Our results provide strong support for the reproductive ground plan hypothesis of evolution in study populations that are independent of the genetic stocks that originally led to the formulation of this hypothesis. As predicted, worker ovary size is genetically linked to multiple correlated traits of the complex division of labor in worker honey bees, known as the pollen hoarding syndrome. The genetic architecture of worker ovary size presumably consists of a combination of trait-specific loci and general regulators that affect the whole behavioral syndrome and may even play a role in caste determination. Several promising candidate genes in the QTL intervals await further study to clarify their potential role in social insect evolution and the regulation of insect fertility in general.

The genetic basis of division of labor in social insects is a central question in evolutionary and behavioral biology. The honey bee is a model for studying evolutionary behavioral genetics because of its well characterized age-correlated division of labor. After an initial period of within-nest tasks, 2-3 week-old worker bees begin foraging outside the nest. Individuals often specialize by biasing their foraging efforts toward collecting pollen or nectar. Efforts to explain the origins of foraging specialization suggest that division of labor between nectar and pollen foraging specialists is influenced by genes with effects on reproductive physiology. Quantitative trait loci (QTL) mapping of foraging behavior also reveals candidate genes for reproductive traits. Here, we address the linkage of reproductive anatomy to behavior, using backcross QTL analysis, behavioral and anatomical phenotyping, candidate gene expression studies, and backcross confirmation of gene-to-anatomical trait associations. Our data show for the first time that the activity of two positional candidate genes for behavior, PDK1 and HR46, have direct genetic relationships to ovary size, a central reproductive trait that correlates with the nectar and pollen foraging bias of workers. These findings implicate two genes that were not known previously to influence complex social behavior. Also, they outline how selection may have acted on gene networks that affect reproductive resource allocation and behavior to facilitate the evolution of social foraging in honey bees.

The effect of larval nutrition on female fertility in honey bees is a focus of both for scientific studies and for practical applications in beekeeping. In general, morphological traits are standards for classifying queens and workers and for evaluating their quality. In recent years, in vitro rearing techniques have been improved and used in many studies; they can produce queen-like and worker-like bees. Here, we questioned whether queens and workers reared in vitro are the same as queens and workers reared in a natural hive environment. We reared workers and queens both in vitro and naturally in beehives to test how these different environments affect metabolic physiology and candidate genes in newly emerged queens and workers. We found that sugar (glucose and trehalose) levels differed between queens and workers in both in vitro and in-hive-reared bees. The in vitro–reared bees had significantly higher levels of lipids in the abdomen. Moreover, hive reared queens had almost 20 times higher levels of vitellogenin than in vitro-reared queens, despite similar morphologies. In addition, hive-reared bees had significantly higher levels of expression of mrjp1. In conclusion, in vitro rearing produces queens and workers that differ from those reared in the hive environment at physiological and gene expression levels.