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1.  Effects of Insemination Quantity on Honey Bee Queen Physiology 
PLoS ONE  2007;2(10):e980.
Mating has profound effects on the physiology and behavior of female insects, and in honey bee (Apis mellifera) queens, these changes are permanent. Queens mate with multiple males during a brief period in their early adult lives, and shortly thereafter they initiate egg-laying. Furthermore, the pheromone profiles of mated queens differ from those of virgins, and these pheromones regulate many different aspects of worker behavior and colony organization. While it is clear that mating causes dramatic changes in queens, it is unclear if mating number has more subtle effects on queen physiology or queen-worker interactions; indeed, the effect of multiple matings on female insect physiology has not been broadly addressed. Because it is not possible to control the natural mating behavior of queens, we used instrumental insemination and compared queens inseminated with semen from either a single drone (single-drone inseminated, or SDI) or 10 drones (multi-drone inseminated, or MDI). We used observation hives to monitor attraction of workers to SDI or MDI queens in colonies, and cage studies to monitor the attraction of workers to virgin, SDI, and MDI queen mandibular gland extracts (the main source of queen pheromone). The chemical profiles of the mandibular glands of virgin, SDI, and MDI queens were characterized using GC-MS. Finally, we measured brain expression levels in SDI and MDI queens of a gene associated with phototaxis in worker honey bees (Amfor). Here, we demonstrate for the first time that insemination quantity significantly affects mandibular gland chemical profiles, queen-worker interactions, and brain gene expression. Further research will be necessary to elucidate the mechanistic bases for these effects: insemination volume, sperm and seminal protein quantity, and genetic diversity of the sperm may all be important factors contributing to this profound change in honey bee queen physiology, queen behavior, and social interactions in the colony.
doi:10.1371/journal.pone.0000980
PMCID: PMC1989138  PMID: 17912357
2.  Chemical Profiles of Two Pheromone Glands Are Differentially Regulated by Distinct Mating Factors in Honey Bee Queens (Apis mellifera L.) 
PLoS ONE  2013;8(11):e78637.
Pheromones mediate social interactions among individuals in a wide variety of species, from yeast to mammals. In social insects such as honey bees, pheromone communication systems can be extraordinarily complex and serve to coordinate behaviors among many individuals. One of the primary mediators of social behavior and organization in honey bee colonies is queen pheromone, which is produced by multiple glands. The types and quantities of chemicals produced differ significantly between virgin and mated queens, and recent studies have suggested that, in newly mated queens, insemination volume or quantity can affect pheromone production. Here, we examine the long-term impact of different factors involved during queen insemination on the chemical composition of the mandibular and Dufour's glands, two of the major sources of queen pheromone. Our results demonstrate that carbon dioxide (an anesthetic used in instrumental insemination), physical manipulation of genital tract (presumably mimicking the act of copulation), insemination substance (saline vs. semen), and insemination volume (1 vs. 8 µl) all have long-term effects on mandibular gland chemical profiles. In contrast, Dufour's gland chemical profiles were changed only upon insemination and were not influenced by exposure to carbon dioxide, manipulation, insemination substance or volume. These results suggest that the chemical contents of these two glands are regulated by different neuro-physiological mechanisms. Furthermore, workers responded differently to the different mandibular gland extracts in a choice assay. Although these studies must be validated in naturally mated queens of varying mating quality, our results suggest that while the chemical composition of Dufour's gland is associated with mating status, that of the mandibular glands is associated with both mating status and insemination success. Thus, the queen appears to be signaling both status and reproductive quality to the workers, which may impact worker behavior and physiology as well as social organization and productivity of the colony.
doi:10.1371/journal.pone.0078637
PMCID: PMC3827242  PMID: 24236028
3.  Queen pheromones in Temnothorax ants: control or honest signal? 
Background
The division of reproductive labor among group members in insect societies is regulated by "queen pheromones". However, it remains controversial whether these are manipulative, i.e., actively suppress worker reproduction, or honestly signal the fertility status of the queen to which workers react in their own interest by refraining from laying eggs. Manipulative queen control is thought to lead to an evolutionary arms race between queens and workers, resulting in complex queen bouquets that diverge strongly among different populations and species. In contrast, honest signals would evolve more slowly and might therefore differ less strongly within and among species.
Results
We aimed at determining the tempo of the evolution of queen signals in two ways. First, we investigated whether queens of Temnothorax ants are capable of controlling egg laying by workers of their own, closely, and distantly related species. Second, we compared the species- and caste-specific patterns of cuticular hydrocarbons, which are assumed to convey information on reproductive status. In mixed-species colonies, queens were not able to fully suppress egg-laying and male production by workers of unrelated species, while workers did not reproduce under the influence of a queen from their own species. Furthermore, the chemical profiles differed more strongly among queens of different species than among the respective workers.
Conclusions
Our results suggest that cuticular hydrocarbons associated with fecundity are not fully conserved in evolution and evolve slightly faster than worker-specific components in the blend of cuticular hydrocarbons. While this higher rate of evolution might reflect an arms race between queens and workers, the observation that workers still respond to the presence of a queen from another species support the honest signal hypothesis. Future studies need to examine alternative explanations for a higher rate of evolution of queen-specific substances, such as an involvement of such compounds in mating.
doi:10.1186/1471-2148-11-55
PMCID: PMC3060118  PMID: 21356125
4.  Genomic analysis of post-mating changes in the honey bee queen (Apis mellifera) 
BMC Genomics  2008;9:232.
Background
The molecular mechanisms underlying the post-mating behavioral and physiological transitions undergone by females have not been explored in great detail. Honey bees represent an excellent model system in which to address these questions because they exhibit a range of "mating states," with two extremes (virgins and egg-laying, mated queens) that differ dramatically in their behavior, pheromone profiles, and physiology. We used an incompletely-mated mating-state to understand the molecular processes that underlie the transition from a virgin to a mated, egg-laying queen. We used same-aged virgins, queens that mated once but did not initiate egg-laying, and queens that mated once and initiated egg-laying.
Results
Differences in the behavior and physiology among groups correlated with the underlying variance observed in the top 50 predictive genes in the brains and the ovaries. These changes were correlated with either a behaviorally-associated pattern or a physiologically-associated pattern. Overall, these results suggest that the brains and the ovaries of queens are uncoupled or follow different timescales; the initiation of mating triggers immediate changes in the ovaries, while changes in the brain may require additional stimuli or take a longer time to complete. Comparison of our results to previous studies of post-mating changes in Drosophila melanogaster identified common biological processes affected by mating, including stress response and alternative-splicing pathways. Comparison with microarray data sets related to worker behavior revealed no obvious correlation between genes regulated by mating and genes regulated by behavior/physiology in workers.
Conclusion
Studying the underlying molecular mechanisms of post-mating changes in honey bee queens will not only give us insight into how molecular mechanisms regulate physiological and behavioral changes, but they may also lead to important insights into the evolution of social behavior. Post-mating changes in gene regulation in the brains and ovaries of honey bee queens appear to be triggered by different stimuli and may occur on different timescales, potentially allowing changes in the brains and the ovaries to be uncoupled.
doi:10.1186/1471-2164-9-232
PMCID: PMC2413142  PMID: 18489784
5.  Queen-signal modulation of worker pheromonal composition in honeybees. 
Worker sterility in honeybees is neither absolute nor irreversible. Whether under queen or worker control, it is likely to be mediated by pheromones. Queen-specific pheromones are not exclusive to queens; workers with activated ovaries also produce them. The association between ovarian activation and queen-like pheromone occurrence suggests the latter as providing a reliable signal of reproductive ability. In this study we investigated the effect of queen pheromones on ovary development and occurrence of queen-like esters in workers' Dufour's gland. Workers separated from the queenright compartment by a double mesh behaved like queenless workers, activating their ovaries and expressing a queen-like Dufour's gland secretion, confirming that the pheromones regulating both systems are non-volatile. Workers with developed ovaries produced significantly more secretion than sterile workers, which we attribute primarily to increased ester production. Workers separated from the queenright compartment by a single mesh displayed a delayed ovarian development, which we attribute to interrupted transfer of the non-volatile pheromone between compartments. We suggest that worker expression of queen-like characters reflects a queen-worker arms race; and that Dufour's gland secretion may provide a reliable signal for ovarian activation. The associative nature between ovary development and Dufour's gland ester production remains elusive.
doi:10.1098/rspb.2004.2839
PMCID: PMC1691821  PMID: 15451697
6.  New insights into honey bee (Apis mellifera) pheromone communication. Is the queen mandibular pheromone alone in colony regulation? 
Frontiers in Zoology  2010;7:18.
Background
In social insects, the queen is essential to the functioning and homeostasis of the colony. This influence has been demonstrated to be mediated through pheromone communication. However, the only social insect for which any queen pheromone has been identified is the honey bee (Apis mellifera) with its well-known queen mandibular pheromone (QMP). Although pleiotropic effects on colony regulation are accredited to the QMP, this pheromone does not trigger the full behavioral and physiological response observed in the presence of the queen, suggesting the presence of additional compounds. We tested the hypothesis of a pheromone redundancy in honey bee queens by comparing the influence of queens with and without mandibular glands on worker behavior and physiology.
Results
Demandibulated queens had no detectable (E)-9-oxodec-2-enoic acid (9-ODA), the major compound in QMP, yet they controlled worker behavior (cell construction and queen retinue) and physiology (ovary inhibition) as efficiently as intact queens.
Conclusions
We demonstrated that the queen uses other pheromones as powerful as QMP to control the colony. It follows that queens appear to have multiple active compounds with similar functions in the colony (pheromone redundancy). Our findings support two hypotheses in the biology of social insects: (1) that multiple semiochemicals with synonymous meaning exist in the honey bee, (2) that this extensive semiochemical vocabulary exists because it confers an evolutionary advantage to the colony.
doi:10.1186/1742-9994-7-18
PMCID: PMC2897789  PMID: 20565874
7.  Queen signals in a stingless bee: suppression of worker ovary activation and spatial distribution of active compounds 
Scientific Reports  2014;4:7449.
In most species of social insect the queen signals her presence to her workers via pheromones. Worker responses to queen pheromones include retinue formation around the queen, inhibition of queen cell production and suppression of worker ovary activation. Here we show that the queen signal of the Brazilian stingless bee Friesella schrottkyi is a mixture of cuticular hydrocarbons. Stingless bees are therefore similar to ants, wasps and bumble bees, but differ from honey bees in which the queen's signal mostly comprises volatile compounds originating from the mandibular glands. This shows that cuticular hydrocarbons have independently evolved as the queen's signal across multiple taxa, and that the honey bees are exceptional. We also report the distribution of four active queen-signal compounds by Matrix-assisted laser desorption/ionization (MALDI) imaging. The results indicate a relationship between the behavior of workers towards the queen and the likely site of secretion of the queen's pheromones.
doi:10.1038/srep07449
PMCID: PMC4264003  PMID: 25502598
8.  Viruses Associated with Ovarian Degeneration in Apis mellifera L. Queens 
PLoS ONE  2011;6(1):e16217.
Queen fecundity is a critical issue for the health of honeybee (Apis mellifera L.) colonies, as she is the only reproductive female in the colony and responsible for the constant renewal of the worker bee population. Any factor affecting the queen's fecundity will stagnate colony development, increasing its susceptibility to opportunistic pathogens. We discovered a pathology affecting the ovaries, characterized by a yellow discoloration concentrated in the apex of the ovaries resulting from degenerative lesions in the follicles. In extreme cases, marked by intense discoloration, the majority of the ovarioles were affected and these cases were universally associated with egg-laying deficiencies in the queens. Microscopic examination of the degenerated follicles showed extensive paracrystal lattices of 30 nm icosahedral viral particles. A cDNA library from degenerated ovaries contained a high frequency of deformed wing virus (DWV) and Varroa destructor virus 1 (VDV-1) sequences, two common and closely related honeybee Iflaviruses. These could also be identified by in situ hybridization in various parts of the ovary. A large-scale survey for 10 distinct honeybee viruses showed that DWV and VDV-1 were by far the most prevalent honeybee viruses in queen populations, with distinctly higher prevalence in mated queens (100% and 67%, respectively for DWV and VDV-1) than in virgin queens (37% and 0%, respectively). Since very high viral titres could be recorded in the ovaries and abdomens of both functional and deficient queens, no significant correlation could be made between viral titre and ovarian degeneration or egg-laying deficiency among the wider population of queens. Although our data suggest that DWV and VDV-1 have a role in extreme cases of ovarian degeneration, infection of the ovaries by these viruses does not necessarily result in ovarian degeneration, even at high titres, and additional factors are likely to be involved in this pathology.
doi:10.1371/journal.pone.0016217
PMCID: PMC3026828  PMID: 21283547
9.  The effects of mating and instrumental insemination on queen honey bee flight behavior and gene expression 
Insect molecular biology  2009;19(2):153-162.
Mating is fundamental to most organisms, although the physiological and transcriptional changes associated with this process have been largely characterized only in Drosophila. In this study, we use honey bees as a model system since their queens undergo massive and permanent physiological and behavioral changes following mating. Previous studies have identified changes associated with the transition from a virgin queen to a fully-mated, egg-laying queen. Here, we further uncouple the mating process to examine the effects of natural mating vs. instrumental insemination and saline vs. semen insemination. We observed effects on flight behavior, vitellogenin expression, and significant overlap in transcriptional profiles between our study and analogous studies in Drosophila, suggesting that some post-mating mechanisms are conserved across insect orders.
doi:10.1111/j.1365-2583.2009.00965.x
PMCID: PMC2989600  PMID: 20002808
10.  Queen pheromones 
Group-living species produce signals that alter the behavior and even the physiology of their social partners. Social insects possess especially sophisticated chemical communication systems that govern every aspect of colony life, including the defining feature of eusociality: reproductive division of labor. Current evidence hints at the central importance of queen pheromones, but progress has been hindered by the fact that such pheromones have only been isolated in honeybees. In a pair of papers on the ant Lasius niger, we identified and investigated a queen pheromone regulating worker sterility. The cuticular hydrocarbon 3-methylhentriacontane (3-MeC31) is correlated with queen maturity and fecundity and workers are also more likely to execute surplus queens that have low amounts of this chemical. Experiments with synthetic 3-MeC31 found that it inhibits ovarian development in queenless workers and lowers worker aggression towards objects coated with it. Production of 3-MeC31 by queens was depressed by an experimental immune challenge, and the same chemical was abundant on queenlaid eggs, suggesting that the workers' responses to the queen are conditional on her health and fecundity. Together with other studies, these results indicate that queen pheromones are honest signals of quality that simultaneously regulate multiple social behaviors.
doi:10.4161/cib.3.6.12976
PMCID: PMC3038062  PMID: 21331238
social insect; queen pheromone; fertility signal; cuticular hydrocarbon; social physiology; primer pheromone
11.  Mate number, kin selection and social conflicts in stingless bees and honeybees 
Microsatellite genotyping of workers from 13 species (ten genera) of stingless bees shows that genetic relatedness is very high. Workers are usually daughters of a single, singly mated queen. This observation, coupled with the multiple mating of honeybee queens, permits kin selection theory to account for many differences in the social biology of the two taxa. First, in contrast to honeybees, where workers are predicted to and do police each other's male production, stingless bee workers are predicted to compete directly with the queen for rights to produce males. This leads to behavioural and reproductive conflict during oviposition. Second, the risk that a daughter queen will attack the mother queen is higher in honeybees, as is the cost of such an attack to workers. This explains why stingless bees commonly have virgin queens in the nest, but honeybees do not. It also explains why in honeybees the mother queen leaves to found a new nest, while in stingless bees it is the daughter queen who leaves.
doi:10.1098/rspb.1999.0648
PMCID: PMC1689682
12.  Sociogenomics of Cooperation and Conflict during Colony Founding in the Fire Ant Solenopsis invicta 
PLoS Genetics  2013;9(8):e1003633.
One of the fundamental questions in biology is how cooperative and altruistic behaviors evolved. The majority of studies seeking to identify the genes regulating these behaviors have been performed in systems where behavioral and physiological differences are relatively fixed, such as in the honey bee. During colony founding in the monogyne (one queen per colony) social form of the fire ant Solenopsis invicta, newly-mated queens may start new colonies either individually (haplometrosis) or in groups (pleometrosis). However, only one queen (the “winner”) in pleometrotic associations survives and takes the lead of the young colony while the others (the “losers”) are executed. Thus, colony founding in fire ants provides an excellent system in which to examine the genes underpinning cooperative behavior and how the social environment shapes the expression of these genes. We developed a new whole genome microarray platform for S. invicta to characterize the gene expression patterns associated with colony founding behavior. First, we compared haplometrotic queens, pleometrotic winners and pleometrotic losers. Second, we manipulated pleometrotic couples in order to switch or maintain the social ranks of the two cofoundresses. Haplometrotic and pleometrotic queens differed in the expression of genes involved in stress response, aging, immunity, reproduction and lipid biosynthesis. Smaller sets of genes were differentially expressed between winners and losers. In the second experiment, switching social rank had a much greater impact on gene expression patterns than the initial/final rank. Expression differences for several candidate genes involved in key biological processes were confirmed using qRT-PCR. Our findings indicate that, in S. invicta, social environment plays a major role in the determination of the patterns of gene expression, while the queen's physiological state is secondary. These results highlight the powerful influence of social environment on regulation of the genomic state, physiology and ultimately, social behavior of animals.
Author Summary
The characterization of the genomic basis for complex behaviors is one of the major goals of biological research. The genomic state of an individual results from the interplay between its internal condition (the “nature”) and the external environment (the “nurture”), which may include the social environment. Colony founding in the fire ant Solenopsis invicta is a complex process that serves as a useful model for investigating how the interplay between genes and social environment shapes social behavior. Unrelated, newly mated S. invicta queens may start a new colony as a group, but ultimately only one queen will survive and gain full reproductive dominance. By uncovering the genetic basis for founding behavior in fire ants we therefore provide useful insights into how cooperative behavior evolved in a context that might be considered primitively eusocial, because newly mated queens in a founding association are morphologically, physiologically and genetically very similar and display no evident division of labor. Our results suggest that social environment (founding singly or in pairs, switching dominance rank vs. maintaining rank) is a much greater driver of gene expression changes than social rank itself, suggesting that social environment, and not reproductive state, is a key regulator of gene expression, physiology and ultimately, behavior.
doi:10.1371/journal.pgen.1003633
PMCID: PMC3738511  PMID: 23950725
13.  The Honey Bee Epigenomes: Differential Methylation of Brain DNA in Queens and Workers 
PLoS Biology  2010;8(11):e1000506.
Using genome-wide methylation profiles in honey bee queen and worker brains to understand how contrasting organismal outputs are generated from the same genotype.
In honey bees (Apis mellifera) the behaviorally and reproductively distinct queen and worker female castes derive from the same genome as a result of differential intake of royal jelly and are implemented in concert with DNA methylation. To determine if these very different diet-controlled phenotypes correlate with unique brain methylomes, we conducted a study to determine the methyl cytosine (mC) distribution in the brains of queens and workers at single-base-pair resolution using shotgun bisulfite sequencing technology. The whole-genome sequencing was validated by deep 454 sequencing of selected amplicons representing eight methylated genes. We found that nearly all mCs are located in CpG dinucleotides in the exons of 5,854 genes showing greater sequence conservation than non-methylated genes. Over 550 genes show significant methylation differences between queens and workers, revealing the intricate dynamics of methylation patterns. The distinctiveness of the differentially methylated genes is underscored by their intermediate CpG densities relative to drastically CpG-depleted methylated genes and to CpG-richer non-methylated genes. We find a strong correlation between methylation patterns and splicing sites including those that have the potential to generate alternative exons. We validate our genome-wide analyses by a detailed examination of two transcript variants encoded by one of the differentially methylated genes. The link between methylation and splicing is further supported by the differential methylation of genes belonging to the histone gene family. We propose that modulation of alternative splicing is one mechanism by which DNA methylation could be linked to gene regulation in the honey bee. Our study describes a level of molecular diversity previously unknown in honey bees that might be important for generating phenotypic flexibility not only during development but also in the adult post-mitotic brain.
Author Summary
The queen honey bee and her worker sisters do not seem to have much in common. Workers are active and intelligent, skillfully navigating the outside world in search of food for the colony. They never reproduce; that task is left entirely to the much larger and longer-lived queen, who is permanently ensconced within the colony and uses a powerful chemical influence to exert control. Remarkably, these two female castes are generated from identical genomes. The key to each female's developmental destiny is her diet as a larva: future queens are raised on royal jelly. This specialized diet is thought to affect a particular chemical modification, methylation, of the bee's DNA, causing the same genome to be deployed differently. To document differences in this epigenomic setting and hypothesize about its effects on behavior, we performed high-resolution bisulphite sequencing of whole genomes from the brains of queen and worker honey bees. In contrast to the heavily methylated human genome, we found that only a small and specific fraction of the honey bee genome is methylated. Most methylation occurred within conserved genes that provide critical cellular functions. Over 550 genes showed significant methylation differences between the queen and the worker, which may contribute to the profound divergence in behavior. How DNA methylation works on these genes remains unclear, but it may change their accessibility to the cellular machinery that controls their expression. We found a tantalizing clue to a mechanism in the clustering of methylation within parts of genes where splicing occurs, suggesting that methylation could control which of several versions of a gene is expressed. Our study provides the first documentation of extensive molecular differences that may allow honey bees to generate different phenotypes from the same genome.
doi:10.1371/journal.pbio.1000506
PMCID: PMC2970541  PMID: 21072239
14.  Ant Queen Egg-Marking Signals: Matching Deceptive Laboratory Simplicity with Natural Complexity 
PLoS ONE  2009;4(3):e4718.
Background
Experiments under controlled laboratory conditions can produce decisive evidence for testing biological hypotheses, provided they are representative of the more complex natural conditions. However, whether this requirement is fulfilled is seldom tested explicitly. Here we provide a lab/field comparison to investigate the identity of an egg-marking signal of ant queens. Our study was based on ant workers resolving conflict over male production by destroying each other's eggs, but leaving queen eggs unharmed. For this, the workers need a proximate cue to discriminate between the two egg types. Earlier correlative evidence indicated that, in the ant Pachycondyla inversa, the hydrocarbon 3,11-dimethylheptacosane (3,11-diMeC27) is more abundant on the surface of queen-laid eggs.
Methodology
We first tested the hypothesis that 3,11-diMeC27 functions as a queen egg-marking pheromone using laboratory-maintained colonies. We treated worker-laid eggs with synthetic 3,11-diMeC27 and found that they were significantly more accepted than sham-treated worker-laid eggs. However, we repeated the experiment with freshly collected field colonies and observed no effect of treating worker-laid eggs with 3,11-diMeC27, showing that this compound by itself is not the natural queen egg-marking pheromone. We subsequently investigated the overall differences of entire chemical profiles of eggs, and found that queen-laid eggs in field colonies are more distinct from worker-laid eggs than in lab colonies, have more variation in profiles, and have an excess of longer-chain hydrocarbons.
Conclusions
Our results suggest that queen egg-marking signals are significantly affected by transfer to the laboratory, and that this change is possibly connected to reduced queen fertility as predicted by honest signaling theory. This change is reflected in the worker egg policing response under field and laboratory conditions.
doi:10.1371/journal.pone.0004718
PMCID: PMC2648039  PMID: 19262683
15.  Expression profiles during honeybee caste determination 
Genome Biology  2000;2(1):research0001.1-research0001.6.
Background
Depending on their larval environment, female honeybees develop into either queens or workers. As in other polyphenisms, this developmental switch depends not on genomic differences between queens and workers but on the differential expression of entire suites of genes involved with larval fate. As such, this and other polyphenic systems can provide a novel tool for understanding how genomes and environmental conditions interact to produce different developmental trajectories. Here we use gene-expression profiles during honeybee caste determination to present the first genomic view of polyphenic development.
Results
Larvae raised as queens or workers differed greatly in their gene-expression patterns. Workers remained more faithful than queens to the expression profiles of younger, bipotential, larvae. Queens appeared to both downregulate many of the genes expressed by bipotential larvae and turn on a distinct set of caste-related genes. Queens overexpressed several metabolic enzymes, workers showed increased expression of a member of the cytochrome P450 family, hexameric storage proteins and dihydrodiol dehydrogenase, and young larvae overexpressed two putative heat-shock proteins (70 and 90 kDa), and several proteins related to RNA processing and translation.
Conclusions
Large differences in gene expression between queens and workers indicate that social insect castes have faced strong directional selection pressures. Overexpression of metabolic enzymes by queen-destined larvae appears to reflect the enhanced growth rate of queens during late larval development. Many of the differently expressed genes we identified have been tied to metabolic rates and cellular responses to hormones, a result consistent with known physiological differences between queen and worker larvae.
PMCID: PMC17597  PMID: 11178278
16.  The transcription factor Krüppel homolog 1 is linked to hormone mediated social organization in bees 
Background
Regulation of worker behavior by dominant queens or workers is a hallmark of insect societies, but the underlying molecular mechanisms and their evolutionary conservation are not well understood. Honey bee and bumble bee colonies consist of a single reproductive queen and facultatively sterile workers. The queens' influences on the workers are mediated largely via inhibition of juvenile hormone titers, which affect division of labor in honey bees and worker reproduction in bumble bees. Studies in honey bees identified a transcription factor, Krüppel-homolog 1 (Kr-h1), whose expression in worker brains is significantly downregulated in the presence of a queen or queen pheromone and higher in forager bees, making this gene an ideal candidate for examining the evolutionary conservation of socially regulated pathways in Hymenoptera.
Results
In contrast to honey bees, bumble bees foragers do not have higher Kr-h1 levels relative to nurses: in one of three colonies levels were similar in nurses and foragers, and in two colonies levels were higher in nurses. Similarly to honey bees, brain Kr-h1 levels were significantly downregulated in the presence versus absence of a queen. Furthermore, in small queenless groups, Kr-h1 levels were downregulated in subordinate workers with undeveloped ovaries relative to dominant individuals with active ovaries. Brain Kr-h1 levels were upregulated by juvenile hormone treatment relative to a vehicle control. Finally, phylogenetic analysis indicates that KR-H1 orthologs are presence across insect orders. Though this protein is highly conserved between honey bees and bumble bees, there are significant differences between orthologs of insects from different orders.
Conclusions
Our results suggest that Kr-h1 is associated with juvenile hormone mediated regulation of reproduction in bumble bees. The expression of this transcription factor is inhibited by the queen and associated with endocrine mediated regulation of social organization in two species of bees. Thus, KR-H1 may transcriptionally regulate a conserved genetic module that is part of a pathway that has been co-opted to function in social behavior, and adjusts the behavior of workers to their social environmental context.
doi:10.1186/1471-2148-10-120
PMCID: PMC2876159  PMID: 20429952
17.  Shared genes related to aggression, rather than chemical communication, are associated with reproductive dominance in paper wasps (Polistes metricus) 
BMC Genomics  2014;15:75.
Background
In social groups, dominant individuals may socially inhibit reproduction of subordinates using aggressive interactions or, in the case of highly eusocial insects, pheromonal communication. It has been hypothesized these two modes of reproductive inhibition utilize conserved pathways. Here, we use a comparative framework to investigate the chemical and genomic underpinnings of reproductive dominance in the primitively eusocial wasp Polistes metricus. Our goals were to first characterize transcriptomic and chemical correlates of reproductive dominance and second, to test whether dominance-associated mechanisms in paper wasps overlapped with aggression or pheromone-related gene expression patterns in other species. To explore whether conserved molecular pathways relate to dominance, we compared wasp transcriptomic data to previous studies of gene expression associated with pheromonal communication and queen-worker differences in honey bees, and aggressive behavior in bees, Drosophila, and mice.
Results
By examining dominant and subordinate females from queen and worker castes in early and late season colonies, we found that cuticular hydrocarbon profiles and genome-wide patterns of brain gene expression were primarily associated with season/social environment rather than dominance status. In contrast, gene expression patterns in the ovaries were associated primarily with caste and ovary activation. Comparative analyses suggest genes identified as differentially expressed in wasp brains are not related to queen pheromonal communication or caste in bees, but were significantly more likely to be associated with aggression in other insects (bees, flies), and even a mammal (mice).
Conclusions
This study provides the first comprehensive chemical and molecular analysis of reproductive dominance in paper wasps. We found little evidence for a chemical basis for reproductive dominance in P. metricus, and our transcriptomic analyses suggest that different pathways regulate dominance in paper wasps and pheromone response in bees. Furthermore, there was a substantial impact of season/social environment on gene expression patterns, indicating the important role of external cues in shaping the molecular processes regulating behavior. Interestingly, genes associated with dominance in wasps were also associated with aggressive behavior in bees, solitary insects and mammals. Thus, genes involved in social regulation of reproduction in Polistes may have conserved functions associated with aggression in insects and other taxa.
doi:10.1186/1471-2164-15-75
PMCID: PMC3922164  PMID: 24472515
Wasps; Social behavior; Genomics; Aggression; Pheromones; Chemical communication
18.  Immune priming and pathogen resistance in ant queens 
Ecology and Evolution  2014;4(10):1761-1767.
Growing empirical evidence indicates that invertebrates become more resistant to a pathogen following initial exposure to a nonlethal dose; yet the generality, mechanisms, and adaptive value of such immune priming are still under debate. Because life-history theory predicts that immune priming and large investment in immunity should be more frequent in long-lived species, we here tested for immune priming and pathogen resistance in ant queens, which have extraordinarily long life span. We exposed virgin and mated queens of Lasius niger and Formica selysi to a low dose of the entomopathogenic fungus Beauveria bassiana, before challenging them with a high dose of the same pathogen. We found evidence for immune priming in naturally mated queens of L. niger. In contrast, we found no sign of priming in virgin queens of L. niger, nor in virgin or experimentally mated queens of F. selysi, which indicates that immune priming in ant queens varies according to mating status and mating conditions or species. In both ant species, mated queens showed higher pathogen resistance than virgin queens, which suggests that mating triggers an up-regulation of the immune system. Overall, mated ant queens combine high reproductive output, very long life span, and elevated investment in immune defense. Hence, ant queens are able to invest heavily in both reproduction and maintenance, which can be explained by the fact that mature queens will be protected and nourished by their worker offspring.
doi:10.1002/ece3.1070
PMCID: PMC4063474  PMID: 24963375
formicine ants; immune priming; immunity; life-history; life span; mating
19.  Comparing Alternative Methods for Holding Virgin Honey Bee Queens for One Week in Mailing Cages before Mating 
PLoS ONE  2012;7(11):e50150.
In beekeeping, queen honey bees are often temporarily kept alive in cages. We determined the survival of newly-emerged virgin honey bee queens every day for seven days in an experiment that simultaneously investigated three factors: queen cage type (wooden three-hole or plastic), attendant workers (present or absent) and food type (sugar candy, honey, or both). Ten queens were tested in each of the 12 combinations. Queens were reared using standard beekeeping methods (Doolittle/grafting) and emerged from their cells into vials held in an incubator at 34C. All 12 combinations gave high survival (90 or 100%) for three days but only one method (wooden cage, with attendants, honey) gave 100% survival to day seven. Factors affecting queen survival were analysed. Across all combinations, attendant bees significantly increased survival (18% vs. 53%, p<0.001). In addition, there was an interaction between food type and cage type (p<0.001) with the honey and plastic cage combination giving reduced survival. An additional group of queens was reared and held for seven days using the best method, and then directly introduced using smoke into queenless nucleus colonies that had been dequeened five days previously. Acceptance was high (80%, 8/10) showing that this combination is also suitable for preparing queens for introduction into colonies. Having a simple method for keeping newly-emerged virgin queens alive in cages for one week and acceptable for introduction into queenless colonies will be useful in honey bee breeding. In particular, it facilitates the screening of many queens for genetic or phenotypic characteristics when only a small proportion meets the desired criteria. These can then be introduced into queenless hives for natural mating or insemination, both of which take place when queens are one week old.
doi:10.1371/journal.pone.0050150
PMCID: PMC3500331  PMID: 23166832
20.  Evolution of thorax architecture in ant castes highlights trade-off between flight and ground behaviors 
eLife  2014;3:e01539.
The concerted evolution of morphological and behavioral specializations has compelling examples in ant castes. Unique to ants is a marked divergence between winged queens and wingless workers, but morphological specializations for behaviors on the ground have been overlooked. We analyzed thorax morphology of queens and workers in species from 21 of the 25 ant subfamilies. We uncovered unique skeletomuscular modifications in workers that presumably increase power and flexibility of head–thorax articulation, emphasizing that workers are not simply wingless versions of queens. We also identified two distinct types of queens and showed repeated evolutionary associations with strategies of colony foundation. Solitary founding queens that hunt have a more worker-like thorax. Our results reveal that ants invest in the relative size of thorax segments according to their tasks. Versatility of head movements allows for better manipulation of food and objects, which arguably contributed to the ants’ ecological and evolutionary success.
DOI: http://dx.doi.org/10.7554/eLife.01539.001
eLife digest
The size and shape of an animal, known as its morphology, often reflect the actions it can perform. A grasshopper’s long legs, for example, are well suited to hopping, whilst the streamlined body of a dolphin helps swimming through water. These specialized features result from the interplay between morphology and behavior during evolution. A change in morphology can make new behaviors possible, which can then expose the animal to new environments and selective pressures that, in turn, can lead to further changes in morphology.
The interplay between morphology and behavior is particularly interesting in social insects such as ants. Queens and workers within an ant colony have a similar set of genes, but they have dramatically different morphologies and very different roles within the colony. Queens are responsible for reproduction, and are larger and have wings, which allow them to fly and establish a new colony away from where they were born. Workers are smaller and lack wings, and they devote themselves to building the nest, feeding the young larvae and protecting the colony. This marked morphological divergence, unique to ants, has fascinated researchers for more than a century. However, most studies have focused on the presence or absence of wings and have overlooked the interactions between morphology and the actions performed on the ground.
Like all insects, an ant’s body is divided into three parts: the head, the thorax (to which the legs and wings are attached), and the abdomen. Now, Keller et al. have examined the shape of the thorax in many species of ants and found that workers are not just smaller wingless versions of queens: rather, the architecture of their thorax is unique among species of flying insects. The front end of the worker thorax is greatly enlarged and is filled by strong neck muscles that power the head and its jaws, and allow workers to hunt and carry prey many times their own weight.
Keller et al. also identified two distinct types of queens and went on to show that these two shapes evolved in association with the two types of strategy that lone queens use to found new colonies. In species where queens convert their own wing muscles into the food for the first generation of workers, the wing muscles are much enlarged and the neck segment is extremely reduced. In species where queens hunt to feed the new colony, the wing and neck muscles are more balanced in size. As such, for those ant species where very little is known about how new colonies are founded, Keller et al. show that we can use the shape of the queen’s thorax to help predict this behavior.
Taken together, the results of Keller et al. show that female ants invest in the relative size of the different segments of the thorax in a way that reflects their behavior as adults. These adaptations partly explain why ants have been so extraordinarily successful in nature, and underscore the importance of carefully analyzing an organism’s form to fully understand its biology.
DOI: http://dx.doi.org/10.7554/eLife.01539.002
doi:10.7554/eLife.01539
PMCID: PMC3881093  PMID: 24399458
ants; Formicidae; social insects; Other
21.  Glandular Epithelium as a Possible Source of a Fertility Signal in Ectatomma tuberculatum (Hymenoptera: Formicidae) Queens 
PLoS ONE  2010;5(4):e10219.
The wax layer covering the insect's cuticle plays an important protective role, as for example, uncontrolled water loss. In social insects, wax production is well-known in some bees that use it for nest building. Curiously, mated-fertile queens of the ant Ectatomma tuberculatum produce an uncommon extra-wax coat and, consequently queens (mated-fertile females) are matte due to such extra cuticular hydrocarbon (CHC) coat that covers the cuticle and masks the brightness of the queens' cuticle while gynes (virgin-infertile queens) are shiny. In this study, histological analysis showed differences in the epidermis between fertile (i.e., queens or gynes with highly ovarian activity) and infertile females (gynes or workers with non developed ovaries). In fertile females the epidermis is a single layer of cubic cells found in all body segments whereas in infertile females it is a thin layer of flattened cells. Ultrastructural features showed active secretory tissue from fertile females similar to the glandular epithelium of wax-producing bees (type I gland). Different hypotheses related to the functions of the glandular epithelium exclusive to the E. tuberculatum fertile queens are discussed.
doi:10.1371/journal.pone.0010219
PMCID: PMC2856672  PMID: 20419093
22.  Identification of an ant queen pheromone regulating worker sterility 
The selective forces that shape and maintain eusocial societies are an enduring puzzle in evolutionary biology. Ordinarily sterile workers can usually reproduce given the right conditions, so the factors regulating reproductive division of labour may provide insight into why eusociality has persisted over evolutionary time. Queen-produced pheromones that affect worker reproduction have been implicated in diverse taxa, including ants, termites, wasps and possibly mole rats, but to date have only been definitively identified in the honeybee. Using the black garden ant Lasius niger, we isolate the first sterility-regulating ant queen pheromone. The pheromone is a cuticular hydrocarbon that comprises the majority of the chemical profile of queens and their eggs, and also affects worker behaviour, by reducing aggression towards objects bearing the pheromone. We further show that the pheromone elicits a strong response in worker antennae and that its production by queens is selectively reduced following an immune challenge. These results suggest that the pheromone has a central role in colony organization and support the hypothesis that worker sterility represents altruistic self-restraint in response to an honest quality signal.
doi:10.1098/rspb.2010.0984
PMCID: PMC2992706  PMID: 20591861
social insect; cuticular hydrocarbon; queen signal; Lasius niger; handicap
23.  Molecular determinants of caste differentiation in the highly eusocial honeybee Apis mellifera 
Background
In honeybees, differential feeding of female larvae promotes the occurrence of two different phenotypes, a queen and a worker, from identical genotypes, through incremental alterations, which affect general growth, and character state alterations that result in the presence or absence of specific structures. Although previous studies revealed a link between incremental alterations and differential expression of physiometabolic genes, the molecular changes accompanying character state alterations remain unknown.
Results
By using cDNA microarray analyses of >6,000 Apis mellifera ESTs, we found 240 differentially expressed genes (DEGs) between developing queens and workers. Many genes recorded as up-regulated in prospective workers appear to be unique to A. mellifera, suggesting that the workers' developmental pathway involves the participation of novel genes. Workers up-regulate more developmental genes than queens, whereas queens up-regulate a greater proportion of physiometabolic genes, including genes coding for metabolic enzymes and genes whose products are known to regulate the rate of mass-transforming processes and the general growth of the organism (e.g., tor). Many DEGs are likely to be involved in processes favoring the development of caste-biased structures, like brain, legs and ovaries, as well as genes that code for cytoskeleton constituents. Treatment of developing worker larvae with juvenile hormone (JH) revealed 52 JH responsive genes, specifically during the critical period of caste development. Using Gibbs sampling and Expectation Maximization algorithms, we discovered eight overrepresented cis-elements from four gene groups. Graph theory and complex networks concepts were adopted to attain powerful graphical representations of the interrelation between cis-elements and genes and objectively quantify the degree of relationship between these entities.
Conclusion
We suggest that clusters of functionally related DEGs are co-regulated during caste development in honeybees. This network of interactions is activated by nutrition-driven stimuli in early larval stages. Our data are consistent with the hypothesis that JH is a key component of the developmental determination of queen-like characters. Finally, we propose a conceptual model of caste differentiation in A. mellifera based on gene-regulatory networks.
doi:10.1186/1471-213X-7-70
PMCID: PMC1929063  PMID: 17577409
24.  Bourgeois queens and high stakes games in the ant Aphaenogaster senilis 
Frontiers in Zoology  2009;6:24.
Background
Many animals face some form of conflict over reproductive opportunities. Queen selection in social insect colonies represents a high-stakes conflict where competition occurs among multiple queens for a few or a single reproductive role(s). The outcome of the contest is critical to the fitness of all colony individuals as most are sterile, and thus represents a conflict at multiple levels. Aphaenogaster senilis is a monogynous, monandrous, fission performing ant, in which queen selection occurs during colony fission and when replacement queens are produced to overcome orphaning. First-born queens are usually behaviourally dominant over subsequent queens, and eventually inherit the colony. We investigated the importance of physical dominance in queen selection in orphaned groups by manipulating the fighting ability of first-born queens via mandibular ablation.
Results
First emerged queens were heavier than second emerged queens, performed almost all aggression, were behaviourally dominant 92% of the time, and prevailed in 76% of groups after co-existing for 16 days on average. Mandibular ablation had no effect on queen behaviour or contest outcome.
Conclusion
Aggression is probably ritualised and contests are decided by workers based on relative queen fertility. First-born queens thus have an inherent advantage over second-born queens as they have more time to develop ovaries. Subordinates never retaliated against aggression from dominants and this lack of retaliation can be interpreted as a form of bourgeois strategy as dominants were almost always first-born. However, the lack of alternative reproductive options makes not-fighting effectively a form of suicide. High relatedness between full-sister queens means that subordinates may be better off sacrificing themselves than risking injury to both queens by fighting.
doi:10.1186/1742-9994-6-24
PMCID: PMC2771002  PMID: 19840383
25.  Genotypic Influence on Aversive Conditioning in Honeybees, Using a Novel Thermal Reinforcement Procedure 
PLoS ONE  2014;9(5):e97333.
In Pavlovian conditioning, animals learn to associate initially neutral stimuli with positive or negative outcomes, leading to appetitive and aversive learning respectively. The honeybee (Apis mellifera) is a prominent invertebrate model for studying both versions of olfactory learning and for unraveling the influence of genotype. As a queen bee mates with about 15 males, her worker offspring belong to as many, genetically-different patrilines. While the genetic dependency of appetitive learning is well established in bees, it is not the case for aversive learning, as a robust protocol was only developed recently. In the original conditioning of the sting extension response (SER), bees learn to associate an odor (conditioned stimulus - CS) with an electric shock (unconditioned stimulus - US). This US is however not a natural stimulus for bees, which may represent a potential caveat for dissecting the genetics underlying aversive learning. We thus first tested heat as a potential new US for SER conditioning. We show that thermal stimulation of several sensory structures on the bee’s body triggers the SER, in a temperature-dependent manner. Moreover, heat applied to the antennae, mouthparts or legs is an efficient US for SER conditioning. Then, using microsatellite analysis, we analyzed heat sensitivity and aversive learning performances in ten worker patrilines issued from a naturally inseminated queen. We demonstrate a strong influence of genotype on aversive learning, possibly indicating the existence of a genetic determinism of this capacity. Such determinism could be instrumental for efficient task partitioning within the hive.
doi:10.1371/journal.pone.0097333
PMCID: PMC4020857  PMID: 24828422

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