Identifying a developmental transition in honey bees using gene expression data.

In many organisms, interactions among genes lead to multiple functional states, and changes to interactions can lead to transitions into new states. These transitions can be related to bifurcations (or critical points) in dynamical systems theory. Characterizing these collective transitions is a major challenge for systems biology. Here, we develop a statistical method for identifying bistability near a continuous transition directly from high-dimensional gene expression data. We apply the method to data from honey bees, where a known developmental transition occurs between bees performing tasks in the nest and leaving the nest to forage. Our method, which makes use of the expected shape of the distribution of gene expression levels near a transition, successfully identifies the emergence of bistability and links it to genes that are known to be involved in the behavioral transition. This proof of concept demonstrates that going beyond correlative analysis to infer the shape of gene expression distributions might be used more generally to identify collective transitions from gene expression data.

A genetic switch for worker nutrition-mediated traits in honeybees.

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.

Genetics of reproduction and regulation of honeybee (Apis mellifera L.) social behavior.

Honeybees form complex societies with a division of labor for reproduction, nutrition, nest construction and maintenance, and defense. How does it evolve? Tasks performed by worker honeybees are distributed in time and space. There is no central control over behavior and there is no central genome on which selection can act and effect adaptive change. For 22 years, we have been addressing these questions by selecting on a single social trait associated with nutrition: the amount of surplus pollen (a source of protein) that is stored in the combs of the nest. Forty-two generations of selection have revealed changes at biological levels extending from the society down to the level of the gene. We show how we constructed this vertical understanding of social evolution using behavioral and anatomical analyses, physiology, genetic mapping, and gene knockdowns. We map out the phenotypic and genetic architectures of food storage and foraging behavior and show how they are linked through broad epistasis and pleiotropy affecting a reproductive regulatory network that influences foraging behavior. This is remarkable because worker honeybees have reduced reproductive organs and are normally sterile; however, the reproductive regulatory network has been co-opted for behavioral division of labor.

Kin selection and eusociality.

Arising from M. A. Nowak, C. E. Tarnita & E. O. Wilson 466, 1057-1062 (2010); Nowak et al. reply. Hamilton described a selective process in which individuals affect kin (kin selection), developed a novel modelling strategy for it (inclusive fitness), and derived a rule to describe it (Hamilton's rule). Nowak et al. assert that inclusive fitness is not the best modelling strategy, and also that its production has been "meagre". The former may be debated by theoreticians, but the latter is simply incorrect. There is abundant evidence to demonstrate that inclusive fitness, kin selection and Hamilton's rule have been extraordinarily productive for understanding the evolution of sociality.

Starvation stress during larval development facilitates an adaptive response in adult worker honey bees (Apis mellifera L.).

Most organisms are constantly faced with environmental changes and stressors. In diverse organisms, there is an anticipatory mechanism during development that can program adult phenotypes. The adult phenotype would be adapted to the predicted environment that occurred during organism maturation. However, whether this anticipatory mechanism is present in eusocial species is questionable because eusocial organisms are largely shielded from exogenous conditions by their stable nest environment. In this study, we tested whether food deprivation during development of the honey bee (Apis mellifera), a eusocial insect model, can shift adult phenotypes to better cope with nutritional stress. After subjecting fifth instar worker larvae to short-term starvation, we measured nutrition-related morphology, starvation resistance, physiology, endocrinology and behavior in the adults. We found that the larval starvation caused adult honey bees to become more resilient toward starvation. Moreover, the adult bees were characterized by reduced ovary size, elevated glycogen stores and juvenile hormone (JH) titers, and decreased sugar sensitivity. These changes, in general, can help adult insects survive and reproduce in food-poor environments. Overall, we found for the first time support for an anticipatory mechanism in a eusocial species, the honey bee. Our results suggest that this mechanism may play a role in honey bee queen-worker differentiation and worker division of labor, both of which are related to the responses to nutritional stress.

Larval starvation improves metabolic response to adult starvation in honey bees (Apis mellifera L.).

Environmental changes during development have long-term effects on adult phenotypes in diverse organisms. Some of the effects play important roles in helping organisms adapt to different environments, such as insect polymorphism. Others, especially those resulting from an adverse developmental environment, have a negative effect on adult health and fitness. However, recent studies have shown that those phenotypes influenced by early environmental adversity have adaptive value under certain (anticipatory) conditions that are similar to the developmental environment, though evidence is mostly from morphological and behavioral observations and it is still rare at physiological and molecular levels. In the companion study, we applied a short-term starvation treatment to fifth instar honey bee larvae and measured changes in adult morphology, starvation resistance, hormonal and metabolic physiology and gene expression. Our results suggest that honey bees can adaptively respond to the predicted nutritional stress. In the present study, we further hypothesized that developmental starvation specifically improves the metabolic response of adult bees to starvation instead of globally affecting metabolism under well-fed conditions. Here, we produced adult honey bees that had experienced a short-term larval starvation, then we starved them for 12 h and monitored metabolic rate, blood sugar concentrations and metabolic reserves. We found that the bees that experienced larval starvation were able to shift to other fuels faster and better maintain stable blood sugar levels during starvation. However, developmental nutritional stress did not change metabolic rates or blood sugar levels in adult bees under normal conditions. Overall, our study provides further evidence that early larval starvation specifically improves the metabolic responses to adult starvation in honey bees.

Genetic architecture of a hormonal response to gene knockdown in honey bees.

Variation in endocrine signaling is proposed to underlie the evolution and regulation of social life histories, but the genetic architecture of endocrine signaling is still poorly understood. An excellent example of a hormonally influenced set of social traits is found in the honey bee (Apis mellifera): a dynamic and mutually suppressive relationship between juvenile hormone (JH) and the yolk precursor protein vitellogenin (Vg) regulates behavioral maturation and foraging of workers. Several other traits cosegregate with these behavioral phenotypes, comprising the pollen hoarding syndrome (PHS) one of the best-described animal behavioral syndromes. Genotype differences in responsiveness of JH to Vg are a potential mechanistic basis for the PHS. Here, we reduced Vg expression via RNA interference in progeny from a backcross between 2 selected lines of honey bees that differ in JH responsiveness to Vg reduction and measured JH response and ovary size, which represents another key aspect of the PHS. Genetic mapping based on restriction site-associated DNA tag sequencing identified suggestive quantitative trait loci (QTL) for ovary size and JH responsiveness. We confirmed genetic effects on both traits near many QTL that had been identified previously for their effect on various PHS traits. Thus, our results support a role for endocrine control of complex traits at a genetic level. Furthermore, this first example of a genetic map of a hormonal response to gene knockdown in a social insect helps to refine the genetic understanding of complex behaviors and the physiology that may underlie behavioral control in general.

New meta-analysis tools reveal common transcriptional regulatory basis for multiple determinants of behavior.

A fundamental problem in meta-analysis is how to systematically combine information from multiple statistical tests to rigorously evaluate a single overarching hypothesis. This problem occurs in systems biology when attempting to map genomic attributes to complex phenotypes such as behavior. Behavior and other complex phenotypes are influenced by intrinsic and environmental determinants that act on the transcriptome, but little is known about how these determinants interact at the molecular level. We developed an informatic technique that identifies statistically significant meta-associations between gene expression patterns and transcription factor combinations. Deploying this technique for brain transcriptome profiles from ca. 400 individual bees, we show that diverse determinants of behavior rely on shared combinations of transcription factors. These relationships were revealed only when we considered complex and variable regulatory rules, suggesting that these shared transcription factors are used in distinct ways by different determinants. This regulatory code would have been missed by traditional gene coexpression or cis-regulatory analytic methods. We expect that our meta-analysis tools will be useful for a broad array of problems in systems biology and other fields.

Regulation of behaviorally associated gene networks in worker honey bee ovaries.

Several lines of evidence support genetic links between ovary size and division of labor in worker honey bees. However, it is largely unknown how ovaries influence behavior. To address this question, we first performed transcriptional profiling on worker ovaries from two genotypes that differ in social behavior and ovary size. Then, we contrasted the differentially expressed ovarian genes with six sets of available brain transcriptomes. Finally, we probed behavior-related candidate gene networks in wild-type ovaries of different sizes. We found differential expression in 2151 ovarian transcripts in these artificially selected honey bee strains, corresponding to approximately 20.3% of the predicted gene set of honey bees. Differences in gene expression overlapped significantly with changes in the brain transcriptomes. Differentially expressed genes were associated with neural signal transmission (tyramine receptor, TYR) and ecdysteroid signaling; two independently tested nuclear hormone receptors (HR46 and ftz-f1) were also significantly correlated with ovary size in wild-type bees. We suggest that the correspondence between ovary and brain transcriptomes identified here indicates systemic regulatory networks among hormones (juvenile hormone and ecdysteroids), pheromones (queen mandibular pheromone), reproductive organs and nervous tissues in worker honey bees. Furthermore, robust correlations between ovary size and neuraland endocrine response genes are consistent with the hypothesized roles of the ovaries in honey bee behavioral regulation.

Complex social behaviour derived from maternal reproductive traits.

A fundamental goal of sociobiology is to explain how complex social behaviour evolves, especially in social insects, the exemplars of social living. Although still the subject of much controversy, recent theoretical explanations have focused on the evolutionary origins of worker behaviour (assistance from daughters that remain in the nest and help their mother to reproduce) through expression of maternal care behaviour towards siblings. A key prediction of this evolutionary model is that traits involved in maternal care have been co-opted through heterochronous expression of maternal genes to result in sib-care, the hallmark of highly evolved social life in insects. A coupling of maternal behaviour to reproductive status evolved in solitary insects, and was a ready substrate for the evolution of worker-containing societies. Here we show that division of foraging labour among worker honey bees (Apis mellifera) is linked to the reproductive status of facultatively sterile females. We thereby identify the evolutionary origin of a widely expressed social-insect behavioural syndrome, and provide a direct demonstration of how variation in maternal reproductive traits gives rise to complex social behaviour in non-reproductive helpers.

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. 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.

Rearing honey bees, Apis mellifera, in vitro 1: effects of sugar concentrations on survival and development.

A new method for rearing honey bees, Apis mellifera L. (Hymenoptera: Apidae), in vitro was developed and the effects of sugar concentrations on survival and development were studied. Seven different glucose (G) and fructose (F) compositions (0%G+0%F, 3%G+3%F, 6%G+6%F, 12%G+12%F, 0%G+12%F, 12%G+0%F, and 4%G+8%F) were tested. Larvae were able to grow to the post defecation stage without addition of sugars (Diet 1), but they were not able to metamorphose and pupate. Adults were reared from diets 2-7. The average larval survival, prepupal larval weights, adult weights, and ovariole numbers were affected significantly due to the sugar compositions in the diets. High sugar concentrations (12%G+12%F) increased the number of queens and intercastes.

Societies to genes: can we get there from here?

Understanding the organization and evolution of social complexity is a major task because it requires building an understanding of mechanisms operating at different levels of biological organization from genes to social interactions. I discuss here, a unique forward genetic approach spanning more than 30 years beginning with human-assisted colony-level selection for a single social trait, the amount of pollen honey bees (Apis mellifera L.) store. The goal was to understand a complex social trait from the social phenotype to genes responsible for observed trait variation. The approach combined the results of colony-level selection with detailed studies of individual behavior and physiology resulting in a mapped, integrated phenotypic architecture composed of correlative relationships between traits spanning anatomy, physiology, sensory response systems, and individual behavior that affect individual foraging decisions. Colony-level selection reverse engineered the architecture of an integrated phenotype of individuals resulting in changes in the social trait. Quantitative trait locus (QTL) studies combined with an exceptionally high recombination rate (60 kb/cM), and a phenotypic map, provided a genotype-phenotype map of high complexity demonstrating broad QTL pleiotropy, epistasis, and epistatic pleiotropy suggesting that gene pleiotropy or tight linkage of genes within QTL integrated the phenotype. Gene expression and knockdown of identified positional candidates revealed genes affecting foraging behavior and confirmed one pleiotropic gene, a tyramine receptor, as a target for colony-level selection that was under selection in two different tissues in two different life stages. The approach presented here has resulted in a comprehensive understanding of the structure and evolution of honey bee social organization.

Hormone response to bidirectional selection on social behavior.

Behavior is a quantitative trait determined by multiple genes. Some of these genes may have effects from early development and onward by influencing hormonal systems that are active during different life-stages leading to complex associations, or suites, of traits. Honey bees (Apis mellifera) have been used extensively in experiments on the genetic and hormonal control of complex social behavior, but the relationships between their early developmental processes and adult behavioral variation are not well understood. Bidirectional selective breeding on social food-storage behavior produced two honey bee strains, each with several sublines, that differ in an associated suite of anatomical, physiological, and behavioral traits found in unselected wild type bees. Using these genotypes, we document strain-specific changes during larval, pupal, and early adult life-stages for the central insect hormones juvenile hormone (JH) and ecdysteroids. Strain differences correlate with variation in female reproductive anatomy (ovary size), which can be influenced by JH during development, and with secretion rates of ecdysteroid from the ovaries of adults. Ovary size was previously assigned to the suite of traits of honey bee food-storage behavior. Our findings support that bidirectional selection on honey bee social behavior acted on pleiotropic gene networks. These networks may bias a bee's adult phenotype by endocrine effects on early developmental processes that regulate variation in reproductive traits.

The gene vitellogenin has multiple coordinating effects on social organization.

Temporal division of labor and foraging specialization are key characteristics of honeybee social organization. Worker honeybees (Apis mellifera) initiate foraging for food around their third week of life and often specialize in collecting pollen or nectar before they die. Variation in these fundamental social traits correlates with variation in worker reproductive physiology. However, the genetic and hormonal mechanisms that mediate the control of social organization are not understood and remain a central question in social insect biology. Here we demonstrate that a yolk precursor gene, vitellogenin, affects a complex suite of social traits. Vitellogenin is a major reproductive protein in insects in general and a proposed endocrine factor in honeybees. We show by use of RNA interference (RNAi) that vitellogenin gene activity paces onset of foraging behavior, primes bees for specialized foraging tasks, and influences worker longevity. These findings support the view that the worker specializations that characterize hymenopteran sociality evolved through co-option of reproductive regulatory pathways. Further, they demonstrate for the first time how coordinated control of multiple social life-history traits can originate via the pleiotropic effects of a single gene that affects multiple physiological processes.

Tyramine and its receptor TYR1 linked behavior QTL to reproductive physiology in honey bee workers (Apis mellifera).

Honey bees (Apis mellifera) provide an excellent model for studying how complex social behavior evolves and is regulated. Social behavioral traits such as the division of labor have been mapped to specific genomic regions in quantitative trait locus (QTL) studies. However, relating genomic mapping to gene function and regulatory mechanism remains a big challenge for geneticists. In honey bee workers, division of labor is known to be regulated by reproductive physiology, but the genetic basis of this regulation remains unknown. In this case, QTL studies have identified tyramine receptor 1 (TYR1) as a candidate gene in region pln2, which is associated with multiple worker social traits and reproductive anatomy. Tyramine (TA), a neurotransmitter, regulates physiology and behavior in diverse insect species including honey bees. Here, we examine directly the effects of TYR1 and TA on worker reproductive physiology, including ovariole number, ovary function and the production of vitellogenin (VG, an egg yolk precursor). First, we used a pharmacology approach to demonstrate that TA affects ovariole number during worker larval development and increases ovary maturation during the adult stage. Second, we used a gene knockdown approach to show that TYR1 regulates vg transcription in adult workers. Finally, we estimated correlations in gene expression and propose that TYR1 may regulate vg transcription by coordinating hormonal and nutritional signals. Taken together, our results suggest TYR1 and TA play important roles in regulating worker reproductive physiology, which in turn regulates social behavior. Our study exemplifies a successful forward-genetic strategy going from QTL mapping to gene function.

Honeybee social regulatory networks are shaped by colony-level selection.

Social interactions pervade all aspects of life in the social insects. Networks of interacting nestmates enable the maintenance of colony homeostasis and regulation of brood development. Artificial colony-level selection on the amount of pollen stored in honeybee colonies has produced high- and low-pollen-hoarding strains that have been used as a model system to study the genetic and physiological basis of differences in forager behavior that contribute to colony-level differences in pollen hoarding. Here we extend this model system using an interacting-phenotypes approach that explicitly studies genetic components arising from social interactions. High- and low-pollen-hoarding-strain larvae were reared in hives with high- or low-strain older larvae and high- or low-strain adult workers. The ovariole number and dry mass of focal individuals depended on interactions between the genotypes of the focal individuals and their brood and adult worker nestmates. These results show that trait expression by individual honeybee workers is modulated by the genotypic composition of the colony, indicating that individual-level phenotypes are properties of the composite "sociogenome." Thus, colony-level selection has produced strains with distinct combinations of socially interacting genes, which make up the social networks that regulate development and expressed phenotypes.

Hierarchical demography: a general approach with an application to honey bees.

Hierarchical population structure, where individuals are aggregated into colonies or similar groups that themselves grow, survive or perish, and potentially produce offspring groups, is an important feature of many biological systems, most notably eusocial organisms such as the honey bee, Apis mellifera. Despite this hierarchical structure, there is a paucity of analytical models and theory linking the dynamics of individuals within colonies to the dynamics of a population of colonies. We present an analytical framework that provides a simple, robust, and predictive theory for the population dynamics of hierarchical organisms. Our framework explicitly describes and links demographic dynamics for the different levels in the hierarchy (individuals, groups, population). We illustrate the application of the framework by developing a model for honey bees and analyzing the effects of life history traits such as worker life span and size at swarming on the growth rate of populations. We conclude by discussing possible extensions of the model that increase its realism and expand its usefulness beyond swarm-founding, monogynous, eusocial insects.

Differences in the morphology, physiology and gene expression of honey bee queens and workers reared in vitro versus in situ.

The effect of larval nutrition on female fertility in honey bees is a focus for both 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.This article has an associated First Person interview with the first author of the paper.

The genetic architecture of sucrose responsiveness in the honeybee (Apis mellifera L.).

One of the best examples of a natural behavioral syndrome is the pollen-hoarding syndrome in honeybees that ties together multiple behavioral phenotypes, ranging from foraging behavior to behavioral ontogeny and learning performance. A central behavioral factor is the bees' responsiveness to sucrose, measured as their proboscis extension reflex. This study examines the genetics of this trait in diploid worker and haploid male honeybees (drones) to learn more about the genetic architecture of the overall behavioral syndrome, using original strains selected for pollen-hoarding behavior. We show that a significant proportion of the phenotypic variability is determined by genotype in males and workers. Second, our data present overwhelming evidence for pleiotropic effects of previously identified quantitative trait loci for foraging behavior (pln-QTL) and epistatic interactions among them. Furthermore, we report on three genomic QTL scans (two reciprocal worker backcrosses and one drone hybrid population) derived from our selection strains. We present at least one significant and two putative new QTL directly affecting the sucrose response of honeybees. Thus, this study demonstrates the modular genetic architecture of behavioral syndromes in general, and elucidates the genetic architecture of the pollen-hoarding behavioral syndrome in particular. Understanding this behavioral syndrome is important for understanding the division of labor in social insects and social evolution itself.