Osnat Malka

Exploring the role of juvenile hormone and vitellogenin in reproduction and social behavior in bumble bees.

BackgroundThe genetic and physiological pathways regulating behavior in solitary species are hypothesized to have been co-opted to regulate social behavior in social species. One classic example is the interaction between vitellogenin (an egg-yolk and storage protein) and juvenile hormone, which are positively correlated in most insect species but have modified interactions in highly eusocial insects. In some of these species (including some termites, ants, and the honey bee), juvenile hormone and vitellogenin levels are negatively correlated and juvenile hormone has shifted its role from a gonadotropin to a regulator of maturation and division of labor in the primarily sterile workers. The function of vitellogenin also seems to have broadened to encompass similar roles. Thus, the functions and molecular interactions of juvenile hormone and vitellogenin are hypothesized to have undergone changes during the evolution of eusociality, but the mechanisms underlying these changes are unknown.Bumble bees offer an excellent model system for testing how the relationship between juvenile hormone and vitellogenin evolved from solitary to social species. Bumble bee colonies are primitively eusocial and comprised of a single reproductive queen and facultatively sterile workers. In Bombus terrestris, juvenile hormone retains its ancestral role as a gonadotropin and is also hypothesized to regulate aggressive behavior. However, the function of vitellogenin and its interactions with juvenile hormone have not yet been characterized.ResultsBy characterizing vitellogenin RNA expression levels (vg) in B. terrestris we show that vg is not associated with task and only partially associated with worker age, queen presence, and caste (queen vs worker). The correlations of vg with ovarian activation were not consistent across experiments, but both vg and ovarian activation were significantly associated with levels of aggression experienced by workers. Treatment with juvenile hormone did not affect vg levels in queenless groups.ConclusionsWe suggest that social interactions affect vg levels more strongly than a worker’s reproductive physiological state, and that juvenile hormone and vg are uncoupled in this species. Thus, although juvenile hormone maintains its traditional role as gonadotropin in B. terrestris, vg has already been co-opted into a novel role, consistent with the model that Bombus represents an intermediate stage in the evolution of eusociality.

Aggressive reproductive competition among hopelessly queenless honeybee workers triggered by pheromone signaling

In the honeybee, Apis mellifera, the queen monopolizes reproduction, while the sterile workers cooperate harmoniously in nest maintenance. However, under queenless (QL) conditions, cooperation collapses and reproductive competition among workers ensues. This is mediated through aggression and worker oviposition, as well as shifts in pheromones, from worker to queen-like composition. Many studies suggest a dichotomy between conflict resolution through aggression or through pheromonal signaling. In this paper, we demonstrate that both phenomena comprise essential components of reproductive competition and that pheromone signaling actually triggers the onset of aggression. We kept workers as QL groups until first aggression was observed and subsequently determined the contestants’ reproductive status and content of the mandibular (MG) and Dufour’s glands (DG). In groups in which aggression occurred early, the attacked bee had consistently more queen-like pheromone in both the MG and DG, although both contestants had undeveloped ovaries. In groups with late aggression, the attacked bee had consistently larger oocytes and more queen-like pheromone in the DG, but not the MG. We suggest that at early stages of competition, the MG secretion is utilized to establish dominance and that the DG provides an honest fertility signal. We further argue that it is the higher amount of DG pheromone that triggers aggression.

The gene road to royalty – differential expression of hydroxylating genes in the mandibular glands of the honeybee

The advances in honeybee sociogenomics have paved the way for the study of social communication processes at the gene level, in particular the expression of caste‐specific pheromones. The queen honeybee mandibular pheromone provides an excellent model system, in that biosynthesis of the hydroxylating fatty acid caste‐specific pheromone appears to be reduced to a single chemical hydroxylation step of stearic acid. Queens are typified by ω‐1‐hydroxylation, as opposed to the worker‐typical ω‐hydroxylation. We hypothesized that this bifurcation is the consequence of differential expression of caste‐specific genes that code for fatty acid‐hydroxylating enzymes from the cytochrome P450 (CYP) family. Bioinformatics studies disclosed two candidate proteins CYP4AA1 and CYP18A1. We thus investigated the expression of these genes in the mandibular glands of queens, and of queenright (QR) and queenless (QL) workers. The real‐time PCR results revealed that CYP4AA1 (ω‐hydroxylation) was expressed at high levels in both QR and QL workers, whereas in queens its expression was negligible. The expression of CYP18A1 (ω‐1‐hydroxylation), on the other hand, was high in the queen’s glands and negligible in those of QR workers. In QL workers, however, the expression of CYP18A1 was considerably elevated and significantly greater than in QR workers. Three‐dimensional structural models constructed for these enzymes demonstrate differences in the active site between CYP18A1 and CYP4AA1, in line with their differential catalytic specificity. The fact that queen pheromone plasticity can be tracked all the way to gene expression provides a new insight into the process of caste differentiation and the accompanying social communication.

Chemical profiles of two pheromone glands are differentially regulated by distinct mating factors in honey bee queens (Apis mellifera L.).

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 Dufours 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, Dufours 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 Dufours 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.

Genomic analysis of the interactions between social environment and social communication systems in honey bees (Apis mellifera)

Social context is often a primary regulator of social behavior, but genes that affect or are affected by social context have rarely been investigated. In social insects, caste specific pheromones are key modulators of social behavior, e.g., in honey bees the queen mandibular gland (MG) pheromone mediates reproductive dominance, its absence prompting ovary activation and queen pheromone production in workers. Here, we investigate the effect of social environment on genome-wide expression patterns in the MG, to determine how social context modulates expression of genes that, in turn alter social environment. We used microarrays to examine the MGs of virgin and mated queens, and queenright (QR) and queenless (QL) workers with or without activated ovaries. Approximately 2554 transcripts were significantly differentially expressed among these groups, with caste and social context being the main regulators of gene expression patterns, while physiological state (ovary activation) only minimally affecting gene expression. Thus, social context strongly regulates expression of genes, which, in turn, shape social environment. Among these, 25 genes that are putatively involved in caste selective production of the fatty-acid derived MG pheromone were differentially expressed in queens and workers. These genes whose functions correspond with enzymatic or transport processes emphasize the occurrence of disparate pheromone biosynthetic pathways for queens and workers, adding another dimension regarding the regulation of these important pheromones. Gene ontology analysis also revealed genes of different functional categories whose expression was impacted by caste or by the social environment, suggesting that the MG has broader functions than pheromone biosynthesis.

Effects of honey bee (Apis mellifera L.) queen insemination volume on worker behavior and physiology

Honey bee colonies consist of tens of thousands of workers and a single reproductive queen that produces a pheromone blend which maintains colony organization. Previous studies indicated that the insemination quantity and volume alter queen mandibular pheromone profiles. In our 11-month long field study we show that workers are more attracted to high-volume versus low-volume inseminated queens, however, there were no significant differences between treatments in the number of queen cells built by workers in preparation for supersedure. Workers exposed to low-volume inseminated queens initiated production of queen-like esters in their Dufours glands, but there were no significant difference in the amount of methyl farnesoate and juvenile hormone in worker hemolymph. Lastly, queen overwintering survival was unexpectedly lower in high-volume inseminated queens. Our results suggest that the queen insemination volume could ultimately affect colony health and productivity.

The role of tyramine and octopamine in the regulation of reproduction in queenless worker honeybees

In honeybees, workers under queenless condition compete for reproduction and establish reproductive dominance hierarchy. Ovary activation is generally accompanied by the expression of queen-like pheromones. Biogenic amines (BAs), in particular dopamine, are believed to be involved in this process by regulating ovarian development. However, the role of BAs in establishing reproductive dominance or their effect on queen-like pheromone production was not investigated. Here, we explored the effect of octopamine (OA) and tyramine (TA) oral treatments on the propensity of treated bees to become reproductively dominant and produce queen-like pheromones in Dufour’s and mandibular glands. One bee in a pair was treated with either OA or TA while the other was fed sugar solution. TA was found to enhance ovary development and the production of esters in the Dufour’s gland and 9HDA (queen component) in the mandibular glands, thus facilitating worker reproductive dominance. OA, on the other hand, did not enhance ovarian development or ester production, but increased the production of 10HDA (worker major component) in the mandibular glands of their sugar-paired mates. OA is known to induce foraging behavior by workers, while increased production of 10HDA characterizes nursing workers. Therefore, we suggest that TA induces reproductive division of labor, while OA treatment results in caste differentiation of workers to foragers and nurses.

Virgin honeybee queens fail to suppress worker fertility but not fertility signalling.

Queen mating status in social insects is a matter of crucial importance for workers because of its influence on the queens productivity and consequently their fitness. Behavioural and physiological reactions of workers to the queens mating status have been studied as a proxy to mechanisms maintaining insect sociality. Here we show that unmated honeybee queens have considerably impaired capacity to trigger worker sterility and cooperative behaviour in comparison to mated (and thus more productive) queens and that under unmated queens social harmony in honeybee societies and queens dominant position are somewhat compromised. Together with this it is shown that honeybee workers exposed to unmated queens despite being active reproductively and behaving accordingly display an impaired ability to advertise their fertility compared to queenless workers. These findings suggest that reproductive development, behavioural reactions and production of fertility signals are differentially regulated and differently influenced by the queens presence.