A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid.
ABSTRACT: By using a functional genomics approach, we have identified a honey bee [Apis mellifera (Am)] odorant receptor (Or) for the queen substance 9-oxo-2-decenoic acid (9-ODA). Honey bees live in large eusocial colonies in which a single queen is responsible for reproduction, several thousand sterile female worker bees complete a myriad of tasks to maintain the colony, and several hundred male drones exist only to mate. The "queen substance" [also termed the queen retinue pheromone (QRP)] is an eight-component pheromone that maintains the queen's dominance in the colony. The main component, 9-ODA, acts as a releaser pheromone by attracting workers to the queen and as a primer pheromone by physiologically inhibiting worker ovary development; it also acts as a sex pheromone, attracting drones during mating flights. However, the extent to which social and sexual chemical messages are shared remains unresolved. By using a custom chemosensory-specific microarray and qPCR, we identified four candidate sex pheromone Ors (AmOr10, -11, -18, and -170) from the honey bee genome based on their biased expression in drone antennae. We assayed the pheromone responsiveness of these receptors by using Xenopus oocytes and electrophysiology. AmOr11 responded specifically to 9-ODA (EC50=280+/-31 nM) and not to any of the other seven QRP components, other social pheromones, or floral odors. We did not observe any responses of the other three Ors to any of the eight QRP pheromone components, suggesting 9-ODA is the only QRP component that also acts as a long-distance sex pheromone.
Project description:Odorant-binding proteins (OBPs) are the critical elements responsible for binding and transporting odors and pheromones in the sensitive olfactory system in insects. Honey bees are representative social insects that have complex odorants and pheromone communication systems relative to solitary insects. Here, we first cloned and characterized OBP11 (AcerOBP11), from the worker bees antennae of Eastern honey bee, Apis cerana. Based on sequence and phylogenetic analysis, most sequences homologous to AcerOBP11 belong to the typical OBPs family. The transcriptional expression profiles showed that AcerOBP11 was expressed throughout the developmental stages and highly specifically expressed in adult antennae. Using immunofluorescence localization, AcerOBP11 in worker bee's antennae was only localized in the sensilla basiconica (SB) near the fringe of each segment. Fluorescence ligand-binding assay showed that AcerOBP11 protein had strong binding affinity with the tested various bee pheromones components, including the main queen mandibular pheromones (QMPs), methyl p-hydroxybenzoate (HOB), and (E)-9-oxo-2-decanoic acid (9-ODA), alarm pheromone (n-hexanol), and worker pheromone components. AcerOBP11 also had strong binding affinity to plant volatiles, such as 4-Allylveratrole. Based on the docking and site-directed mutagenesis, two key amino acid residues (Ile97 and Ile140) were involved in the binding of AcerOBP11 to various bee pheromones. Taken together, we identified that AcerOBP11 was localized in a single type of antennal chemosensilla and had complex ligand-binding properties, which confer the dual-role with the primary characteristics of sensing various bee pheromones and secondary characteristics of sensing general odorants. This study not only prompts the theoretical basis of OBPs-mediated bee pheromones recognition of honey bee, but also extends the understanding of differences in pheromone communication between social and solitary insects.
Project description:During emergency queen rearing, worker honey bees (Apis mellifera) select several otherwise worker-destined larvae to instead rear as candidates to replace their dead or failing queen. This choice is crucial as the queen is the sole reproductive in the colony and her quality is essential to its success. Because honey bee queens mate with and store sperm from multiple drones, emergency queen selection presents workers with an opportunity to increase fitness by selecting full- (0.75 relatedness), rather than half- (0.25 relatedness), sisters as new queen candidates. Through patriline analysis of colonies along with large numbers of emergency queens reared by each we affirm the purported "royal" patriline theory that, instead of competing nepotistically, workers exhibit bias towards selecting individuals from particular "royal" subfamilies during emergency queen rearing events, Further, we show that these "royal" patrilines are cryptic in honey bee colonies; occurring in such low frequency in the overall colony population that they are frequently undetected in traditional tests of queen mating number and colony composition. The identification of these cryptic "royal" subfamilies reveals that honey bee queens, already considered "hyperpolyandrous," are mating with even more males than has been previously recognized. These results alter our understanding of reproductive behavior in honey bees, raising questions about the evolutionary implications of this phenomenon.
Project description: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.
Project description:Responses to social cues, such as pheromones, can be modified by genotype, physiology, or environmental context. Honey bee queens produce a pheromone (queen mandibular pheromone; QMP) which regulates many aspects of worker bee behavior and physiology. Forager honey bees are less responsive to QMP than young nurse bees engaged in brood care, suggesting that physiological changes associated with behavioral maturation may modulate response to this pheromone. Since cGMP is a major regulator of behavioral maturation in honey bee workers, we examined its role in modulating worker responses to QMP. Treatment with a cGMP analog, 8-Br-cGMP, resulted in significant reductions in both behavioral and physiological responses to QMP in young caged workers. Treatment significantly reduced attraction to QMP (the retinue response) and inhibited the QMP-mediated increase in vitellogenin levels in the fat bodies of worker bees. Genome-wide analysis of brain gene expression patterns demonstrated that cGMP has a larger effect on expression levels than QMP, and that QMP has specific effects in the presence of cGMP, suggesting that some responses to QMP may be dependent on an individual beesM-^R physiological state. Several functional gene categories were significantly differentially expressed, including genes involved in regulating GTPase activity, phototransduction, immunity, and carboxylic acid transmembrane transporter activity. Overall, our data suggest that cGMP-mediated processes play a large role in modulating responses to queen pheromone in honey bees, at the behavioral, physiological and molecular levels.
Project description:Olfactory/odorant receptors (ORs) probably govern eusocial behaviour in honey bees through detection of cuticular hydrocarbons (CHCs) and queen mandibular gland pheromones (QMP). CHCs are involved in nest-mate recognition whereas QMP acts as sex pheromone for drones and as retinue pheromone for female workers. Further studies on the effect of eusociality on the evolution of ORs are hindered by the non-availability of comprehensive OR sets of solitary species. We report complete OR repertoires from two solitary bees Dufourea novaeangliae (112 ORs) and Habropoda laboriosa (151 ORs). We classify these ORs into 34 phylogenetic clades/subfamilies. Differences in the OR sets of solitary and eusocial bees are observed in individual subfamilies like subfamily 9-exon (putative CHC receptors) and L (contains putative QMP receptor group). A subfamily (H) including putative floral scent receptors is expanded in the generalist honey bees only, but not in the specialists. On the contrary, subfamily J is expanded in all bees irrespective of their degree of social complexity or food preferences. Finally, we show species-lineage specific and OR-subfamily specific differences in the putative cis-regulatory DNA motifs of the ORs from six hymenopteran species. Out of these, [A/G]CGCAAGCG[C/T] is a candidate master transcription factor binding site for multiple olfactory genes.
Project description:The Asian hornet, Vespa velutina, is an invasive, globally-distributed predator of European honey bees and other insects. To better under its reproductive biology and to find a specific, effective, and low-impact control method for this species, we identified and tested the key compounds in V. velutina sex pheromone. Virgin gynes (reproductive females) produced this sex pheromone in the sixth intersegmental sternal glands of their abdomens. The active compounds were 4-oxo-octanoic acid (4-OOA, 10.4??g bee<sup>-1</sup>) and 4-oxo-decanoic acid (4-ODA, 13.3??g bee<sup>-1</sup>) at a 0.78 ratio of 4-OOA/4-ODA. We synthesized these compounds and showed that male antennae were highly sensitive to them. Moreover, males were only strongly attracted to a 4-OOA/4-ODA blend at the natural ratio produced by gynes. These results provide the first demonstration of an effective way to lure V. velutina males, and the first chemical identification of a sex pheromone in the eusocial hornets.
Project description: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.
Project description:Responses to social cues, such as pheromones, can be modified by genotype, physiology, or environmental context. Honey bee queens produce a pheromone (queen mandibular pheromone; QMP) which regulates many aspects of worker bee behavior and physiology. Forager honey bees are less responsive to QMP than young nurse bees engaged in brood care, suggesting that physiological changes associated with behavioral maturation may modulate response to this pheromone. Since cGMP is a major regulator of behavioral maturation in honey bee workers, we examined its role in modulating worker responses to QMP. Treatment with a cGMP analog, 8-Br-cGMP, resulted in significant reductions in both behavioral and physiological responses to QMP in young caged workers. Treatment significantly reduced attraction to QMP (the retinue response) and inhibited the QMP-mediated increase in vitellogenin levels in the fat bodies of worker bees. Genome-wide analysis of brain gene expression patterns demonstrated that cGMP has a larger effect on expression levels than QMP, and that QMP has specific effects in the presence of cGMP, suggesting that some responses to QMP may be dependent on an individual bees physiological state. Several functional gene categories were significantly differentially expressed, including genes involved in regulating GTPase activity, phototransduction, immunity, and carboxylic acid transmembrane transporter activity. Overall, our data suggest that cGMP-mediated processes play a large role in modulating responses to queen pheromone in honey bees, at the behavioral, physiological and molecular levels.
Project description:Honey bee (Apis mellifera) queens have a remarkable organ, the spermatheca, which successfully stores sperm for years after a virgin queen mates. This study uniquely characterized and quantified the transcriptomes of the spermathecae from mated and virgin honey bee queens via RNA sequencing to identify differences in mRNA levels based on a queen's mating status. The transcriptome of drone semen was analyzed for comparison. Samples from three individual bees were independently analyzed for mated queen spermathecae and virgin queen spermathecae, and three pools of semen from ten drones each were collected from three separate colonies. In total, the expression of 11,233 genes was identified in mated queen spermathecae, 10,521 in virgin queen spermathecae, and 10,407 in drone semen. Using a cutoff log2 fold-change value of 2.0, we identified 212 differentially expressed genes between mated and virgin spermathecal queen tissues: 129 (1.4% of total) were up-regulated and 83 (0.9% of total) were down-regulated in mated queen spermathecae. Three genes in mated queen spermathecae, three genes in virgin queen spermathecae and four genes in drone semen that were more highly expressed in those tissues from the RNA sequencing data were further validated by real time quantitative PCR. Among others, expression of Kielin/chordin-like and Trehalase mRNAs was highest in the spermathecae of mated queens compared to virgin queen spermathecae and drone semen. Expression of the mRNA encoding Alpha glucosidase 2 was higher in the spermathecae of virgin queens. Finally, expression of Facilitated trehalose transporter 1 mRNA was greatest in drone semen. This is the first characterization of gene expression in the spermathecae of honey bee queens revealing the alterations in mRNA levels within them after mating. Future studies will extend to other reproductive tissues with the purpose of relating levels of specific mRNAs to the functional competence of honey bee queens and the colonies they head.