Gene co-citation networks associated with worker sterility in honey bees.

BACKGROUND: The evolution of reproductive self-sacrifice is well understood from kin theory, yet our understanding of how actual genes influence the expression of reproductive altruism is only beginning to take shape. As a model in the molecular study of social behaviour, the honey bee Apis mellifera has yielded hundreds of genes associated in their expression with differences in reproductive status of females, including genes directly associated with sterility, yet there has not been an attempt to link these candidates into functional networks that explain how workers regulate sterility in the presence of queen pheromone. In this study we use available microarray data and a co-citation analysis to describe what gene interactions might regulate a worker\u27s response to ovary suppressing queen pheromone. RESULTS: We reconstructed a total of nine gene networks that vary in size and gene composition, but that are significantly enriched for genes of reproductive function. The networks identify, for the first time, which candidate microarray genes are of functional importance, as evidenced by their degree of connectivity to other genes within each of the inferred networks. Our study identifies single genes of interest related to oogenesis, including eggless, and further implicates pathways related to insulin, ecdysteroid, and dopamine signaling as potentially important to reproductive decision making in honey bees. CONCLUSIONS: The networks derived here appear to be variable in gene composition, hub gene identity, and the overall interactions they describe. One interpretation is that workers use different networks to control personal reproduction via ovary activation, perhaps as a function of age or environmental circumstance. Alternatively, the multiple networks inferred here may represent segments of the larger, single network that remains unknown in its entirety. The networks generated here are provisional but do offer a new multi-gene framework for understanding how honey bees regulate personal reproduction within their highly social breeding system

Understanding honey bee worker sterility: a conceptual-empirical framework

Kin selection explains how complex social behaviour can evolve at the gene level, but this theory does not identify which genes are necessary for the expression of altruism. In my first chapter I overview seven criteria for genes for altruism using the honey bee as a model species. In the second chapter I explore one criterion in detail – that altruism genes are differentially expressed between reproductive and sterile workers. I used results from previous microarray studies to reconstruct nine knowledge-based gene networks that describe reproductive altruism by means of ovary activation and de-activation. All networks were enriched for Gene Ontology terms pertaining to reproduction, and the hub genes in each network tend to consist of genes involved in expression and signaling. 138 genes overlap among networks for workers of different ages and tissues. These networks provide testable hypotheses that explain the expression of altruistic sterility in workers

Light up the fly: Drosophila as a non-social model in insect sociobiology

Eusocial breeding systems are characterized by a reproductive division of labour. For many social taxa, the queen signals her fecundity to her daughters via a pheromone, which renders them sterile. Solitary insects, in contrast, lack social organization and their personal reproduction is not regulated by social cues. Despite these radically different breeding habits between these two taxa, one prediction from sociogenomic theory is that eusocial taxa evolved their complex caste system through co-option of pathways already present in solitary ancestors. In this thesis, I present a series of comparative experiments that provide support for these conserved genes and gene pathways that regulate reproduction in social versus non-social taxa. First, I show that distinctly non-social Drosophila melanogaster can respond to a highly social Apis mellifera pheromone (QMP) in a manner similar to sterile worker bees – namely, by turning off their ovaries and foregoing reproduction. Second, I show that this conspicuous interspecific response is conserved at a genetic level, where the presence of certain foraging alleles can elicit variable responses to the pheromone in a manner similar to that in the bee. Third, I suggest that solitary and eusocial species use a conserved olfactory signaling mechanism to elicit reproductive responses to QMP. Using mutant Drosophila lines and an RNAi-mediated screen of olfactory receptors, I identify five top receptors as candidates for the perception of QMP and subsequent reduced ovary phenotypes. Lastly, I use Drosophila to investigate the functional association between two opposing social cues, royal jelly and QMP and their ability to modulate ovarian development. These results showcase the power of the comparative approach in identifying genes and gene pathways involved in the regulation of worker sterility, and suggest that the genetic basis of characteristically eusocial behaviours like reproductive altruism, are conserved in non-social insects

Structure and function of gene regulatory networks associated with worker sterility in honeybees.

A characteristic of eusocial bees is a reproductive division of labor in which one or a few queens monopolize reproduction, while her worker daughters take on reproductively altruistic roles within the colony. The evolution of worker reproductive altruism involves indirect selection for the coordinated expression of genes that regulate personal reproduction, but evidence for this type of selection remains elusive. In this study, we tested whether genes coexpressed under queen-induced worker sterility show evidence of adaptive organization within a model brain transcriptional regulatory network (TRN). If so, this structured pattern would imply that indirect selection on nonreproductive workers has influenced the functional organization of genes within the network, specifically to regulate the expression of sterility. We found that literature-curated sets of candidate genes for sterility, ranging in size from 18 to 267, show strong evidence of clustering within the three-dimensional space of the TRN. This finding suggests that our candidate sets of genes for sterility form functional modules within the living bee brain\u27s TRN. Moreover, these same gene sets colocate to a single, albeit large, region of the TRN\u27s topology. This spatially organized and convergent pattern contrasts with a null expectation for functionally unrelated genes to be haphazardly distributed throughout the network. Our meta-genomic analysis therefore provides first evidence for a truly social transcriptome that may regulate the conditional expression of honeybee worker sterility

Genetic components to worker sterility in the honey bee, Apis mellifera

The primary characteristic that defines eusocial species is reproductive division of labour. Honey bee (Apis mellifera) colonies typically have a single reproductive queen and thousands of sterile workers. Here, I review the factors affecting worker reproduction and then contrast the brain gene expression of workers considered either reproductively altruistic (sterile) or selfish (fecund) over a series of time points. I confirmed that although, theoretically, the genes that allow workers to reproduce must be expressed in order for them to do so, it is the environmental cues, such as nutrition and pheromones, that ultimately control worker reproductive status. I then identify a new set of candidate ‘genes for reproductive altruism’ by considering the differential gene expression of reproductive vs. sterile worker brains on each day, and over multiple consecutive time-points. It was determined that a large portion of the identified genes had metabolic function

Drosophila as a genetically tractable model for social insect behavior

The relatively simple communication, breeding, and egg-making systems that govern reproduction in female Drosophila retain homology to eusocial species in which these same systems are modified to the social condition. Despite having no parental care, division of labor, or subfertile caste, Drosophila may nonetheless offer a living test of certain sociobiological hypotheses framed around gene function. In this review, we make this case, and do so around the recent discovery that the non-social fly, Drosophila melanogaster, can respond to the ovary-suppressing queen pheromone of the honey bee Apis mellifera. Here, we first explain the sociobiological imperative to reconcile kin theory with molecular biology, and qualify a potential role for Drosophila. Then, we offer three applications for the fly-pheromone assay. First, the availability and accessibility of massive mutant libraries makes immediately feasible any number of open or targeted gene screens against the ovary-inhibiting response. The sheer tractability of Drosophila may therefore help to accelerate the search for genes in pheromone-responsive pathways that regulate female reproduction, including potentially any that are preserved with modification to regulate worker sterility in response to queen pheromones in eusocial taxa. Secondly, Drosophila's powerful Gal4/UAS expression system can complement the pheromone assay by driving target gene expression into living tissue, which could be well-applied to the functional testing of genes presumed to drive ovary activation or de-activation in the honey bee or other eusocial taxa. Finally, coupling Gal4 with UAS-RNAi lines can facilitate loss-of-function experiments against perception and response to the ovary inhibiting pheromone, and do so for large numbers of candidates in systematic fashion. Drosophila's utility as an adjunct to the field of insect sociobiology is not ideal, but retains surprising potential

Sensory Response System of Social Behavior Tied to Female Reproductive Traits

Honey bees display a complex set of anatomical, physiological, and behavioral traits that correlate with the colony storage of surplus pollen (pollen hoarding). We hypothesize that the association of these traits is a result of pleiotropy in a gene signaling network that was co-opted by natural selection to function in worker division of labor and foraging specialization. By acting on the gene network, selection can change a suite of traits, including stimulus/response relationships that affect individual foraging behavior and alter the colony level trait of pollen hoarding. The 'pollen-hoarding syndrome' of honey bees is the best documented syndrome of insect social organization. It can be exemplified as a link between reproductive anatomy (ovary size), physiology (yolk protein level), and foraging behavior in honey bee strains selected for pollen hoarding, a colony level trait. The syndrome gave rise to the forager-Reproductive Ground Plan Hypothesis (RGPH), which proposes that the regulatory control of foraging onset and foraging preference toward nectar or pollen was derived from a reproductive signaling network. This view was recently challenged. To resolve the controversy, we tested the associations between reproductive anatomy, physiology, and stimulus/response relationships of behavior in wild-type honey bees.Central to the stimulus/response relationships of honey bee foraging behavior and pollen hoarding is the behavioral trait of sensory sensitivity to sucrose (an important sugar in nectar). To test the linkage of reproductive traits and sensory response systems of social behavior, we measured sucrose responsiveness with the proboscis extension response (PER) assay and quantified ovary size and vitellogenin (yolk precursor) gene expression in 6-7-day-old bees by counting ovarioles (ovary filaments) and by using semiquantitative real time RT-PCR. We show that bees with larger ovaries (more ovarioles) are characterized by higher levels of vitellogenin mRNA expression and are more responsive to sucrose solutions, a trait that is central to division of labor and foraging specialization.Our results establish that in wild-type honey bees, ovary size and vitellogenin mRNA level covary with the sucrose sensory response system, an important component of foraging behavior. This finding validates links between reproductive physiology and behavioral-trait associations of the pollen-hoarding syndrome of honey bees, and supports the forager-RGPH. Our data address a current evolutionary debate, and represent the first direct demonstration of the links between reproductive anatomy, physiology, and behavioral response systems that are central to the control of complex social behavior in insects

Tracking behavioural and neuronal responses to social pheromones: Insights from a Drosophila model

If eusociality evolved through modification of pre-social mechanisms for regulating personal reproduction, then even insects like Drosophila may be vulnerable to latent effects of \u27queen\u27 pheromone. Here, I test if male fruit flies respond to a eusocial queen bee pheromone. I found that male flies were attracted to queen bee pheromone, and pheromone-treated males raised the intensity of their courting towards conspecific females. These novel observations from Drosophila suggest that male flies have the capacity to respond to queen pheromone in a manner that is comparable to the native response from male (drone) bees. I therefore optimized a nuclear factor of activated T-cell (NFAT) system to label olfactory neurons that are putatively responsive to the pro-reproductive pheromone. The NFAT reporter system implicates three neurons (Or-49b, Or-56a, Or-98a) that, if shown to function similarly in drones, will validate my use of Drosophila to probe otherwise unknown mechanisms of social bee communication

Differential Proteomics in Dequeened Honeybee Colonies Reveals Lower Viral Load in Hemolymph of Fertile Worker Bees

The eusocial societies of honeybees, where the queen is the only fertile female among tens of thousands sterile worker bees, have intrigued scientists for centuries. The proximate factors, which cause the inhibition of worker bee ovaries, remain largely unknown; as are the factors which cause the activation of worker ovaries upon the loss of queen and brood in the colony. In an attempt to reveal key players in the regulatory network, we made a proteomic comparison of hemolymph profiles of workers with completely activated ovaries vs. rudimentary ovaries. An unexpected finding of this study is the correlation between age matched worker sterility and the enrichment of Picorna-like virus proteins. Fertile workers, on the other hand, show the upregulation of potential components of the immune system. It remains to be investigated whether viral infections contribute to worker sterility directly or are the result of a weaker immune system of sterile workers