Erika Plettner

Mandibular gland components and ovarian development as measures of caste differentiation in the honey bee (Apis mellifera L.)

Abstract Changes in mandibular gland pheromone production and ovarian development were investigated in queenless North American honey bees (Apis mellifera L.). An intercaste, false queens, laying workers, queenless foragers and queenright workers were analyzed for the amounts of five mandibular gland pheromone components and for ovarian development. Pheromone composition was not related to ovarian development, in contrast to some previous studies with the African race, Apis mellifera capensis. These results suggest that queen/worker caste differentiation may be more strongly fixed during development in some honey bee races than in others.

Mandibular gland components of european and africanized honey bee queens (Apis mellifera L.).

The composition of the five-component honey bee queen mandibular gland pheromone (QMP) of mated European honey bee queens was compared to those of virgin and drone-laying (i.e., laying only haploid unfertilized eggs that develop into males), European queens and Africanized mated queens. QMP of mated European queens showed significantly greater quantities of individual components than all queen types compared, except for a significantly greater quantity of 9-hydroxy-(E)-2-decenoic acid (9-HDA) found in Africanized queens. Glands of European drone-laying queens contained quantities intermediate between virgin and mated queens, reflecting their intermediate reproductive state and age. QMP ontogeny shifts from a high proportion of 9-keto-(E)-2-decenoic acid (ODA) in young unmated queens to roughly equal proportions of ODA and 9-HDA in mated queens. A biosynthetic shift occurs after mating that results in a greater proportion of 9-HDA, methylp-hydroxybenzoate (HOB), and 4-hydroxy-3-methoxyphenylethanol (HVA) production, accompanied by a decreased proportion of ODA. Africanized QMP proportions of ODA and 9-HDA were significantly different from European queens. A quantitative definition of a “queen equivalent” of QMP is proposed for the various queen types, and a standard queen equivalent for mated European honeybee queen mandibular gland pheromone is adopted as 200µg ODA, 80µg 9-HDA, 20µg HOB, and 2 µg HVA.

Effects of social environment and worker mandibular glands on endocrine-mediated behavioral development in honey bees

Abstract Previous studies suggest that older honey bee workers possess an inhibitory signal that regulates behavioral development in younger bees. To study how this inhibitor is transmitted, bees were reared for 7 days in double-screen cages, single-screen cages, or unrestricted in a typical colony (control bees). Double-screen cages prevented physical contact with colony members while single-screen cages allowed only antennation and food exchange. Bees reared in double-screen cages showed accelerated endocrine and behavioral development; they had significantly higher rates of juvenile hormone biosynthesis and juvenile hormone titers than did control bees and also were more likely to become precocious foragers. Relative to the other two groups, bees reared in single-screen cages showed intermediate juvenile hormone biosynthesis rates and titers, and intermediate rates of behavioral development. These results indicate that physical contact is required for total inhibition. We also began to test the hypothesis that worker mandibular glands are the sources of an inhibitory signal. Old bees with mandibular glands removed were significantly less inhibitory towards young bees than were sham-operated and unoperated bees. These results suggest that an inhibitor is produced by the worker mandibular glands.

Biosynthesis of Mandibular Acids in Honey Bees (Apis mellifera): De novo Synthesis, Route of Fatty Acid Hydroxylation and Caste Selective β-Oxidation

Abstract Fatty acids functionalized at the last (ω) and penultimate (ω-1) position, found in the mandibular glands of worker and queen honey bees (Apis mellifera L.), have important functions in the colony and are caste specific. Queens have predominantly 10-carbon ω-1-functionalized acids and workers have 10-carbon ω-functionalized acids. In previous work we have shown that the mandibular acids are synthesized from octadecanoic acid in three steps: (1) hydroxylation at the ω and ω-1 position; (2) β-oxidation of the 18-carbon hydroxy acids to the 8 and 10-carbon length; and (3) oxidation of the ω- and ω-1-hydroxy groups to give diacids and 9-keto-2(E)-decenoic acid, respectively. The last two steps are caste selective. In this work, we studied the biosynthesis of mandibular acids from acetate, distinguished among two possible routes of hydroxylation and studied caste differences in hydroxy acid chain shortening. Workers glands biosynthesize mandibular acids from acetate and, therefore, do not depend on an external source of octadecanoic acid. Hydroxylation at the ω position proceeds with retention of label at the ω-1 position. Hydroxylation at the ω-1 position proceeds with retention of label at the ω position and does not involve a terminal double bond. Finally, hydroxy acid chain shortening differs in queens and workers in two respects: (1) the inhibition pattern in the presence of 2-fluorooctadecanoic acid; and (2) reversibility. Chain shortening is inhibited by 2-fluorooctadecanoic acid to a greater extent in workers than in queens. Furthermore, workers are able to elongate hydroxy acids to the next higher 2-carbon homologue and are able to reduce hydroxy-2(E)-decenoic acids to the corresponding hydroxydecanoic acids. These transformations were not detected in queens.

A scientific note on E-β -ocimene, a new volatile primer pheromone that inhibits worker ovary development in honey bees

In social insects, larvae are completely dependent on adults to complete their development. In honey bees (Apis mellifera L.), larval development requires that larvae engage workers in nursing tasks rather than allocating energy in other activities (Le Conte and Hefetz, 2008). Indeed, in the absence of any constraint, workers tend to become active reproductive individual. The queen inhibits worker ovary development via pheromones (Hoover et al., 2003; Wossler and Crewe, 1999), but the inhibition of worker ovary development seems superior when bees are exposed to unsealed brood (Kropacova and Haslbachova, 1970, 1971). Components of the brood pheromone have already been identified and consist of a blend of 10 esters (Le Conte et al., 1989, 1990). Two of these low volatility compounds have been demonstrated to partially decrease worker ovary development (Mohammedi et al., 1998; Pankiw and Garza, 2007). Thus, we asked whether brood emits volatile compounds that could also have an effect on worker ovary activation. We identified a new highly volatile molecule from the larvae E-β-ocimene, that inhibits worker ovary maturation. We used Solid Phase Microextraction (SPME) (65 μm Carbowax) to sample volatiles emitted by different larval instars. We analyzed 20 larvae at stage 1, 2–3 or 10 larvae at stage 4–5. Larvae were kept in a 15 mL closed vial for 20 min in an incubator at 34 ◦C and 50% humidity (N = 8,

Nosema spp. Infection Alters Pheromone Production in Honey Bees (Apis mellifera)

Pheromones in social insects play a key role in the regulation of group homoeostasis. It is well-established that parasites can modify hormone signaling of their host, but less is known about the effect of parasites on pheromone signaling in insect societies. We, thus, tested in honey bees (Apis mellifera) the effect of the widespread parasite Nosema spp. on the production of ethyl oleate (EO), the only identified primer pheromone in honey bee workers. Since environmental stressors like pesticides also can weaken honey bees, we also analyzed the effect of imidacloprid, a neonicotinoid widely used in agriculture, on EO production. We show that, contrary to imidacloprid, Nosema spp. significantly altered EO production. In addition, the level of Nosema infection was correlated positively with the level of EO production. Since EO is involved in the regulation of division of labor among workers, our result suggests that the changes in EO signaling induced by parasitism have the potential to disturb the colony homoeostasis.

New insights into honey bee (Apis mellifera) pheromone communication. Is the queen mandibular pheromone alone in colony regulation

BackgroundIn 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.ResultsDemandibulated 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.ConclusionsWe 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.

A combinatorial approach to chemical modification of subtilisin Bacillus lentus

The reaction between methanethiosulfonate reagents and cysteine mutants of subtilisin is quantitative and can be used to prepare chemically modified mutant enzymes (CMMs) with novel properties. The virtually unrestricted structural variations possible for CMMs presents a preparative and screening challenge. To address this, a rapid combinatorial method for preparing and screening the activities of CMMs has been developed.

Seasonal variation in the titers and biosynthesis of the primer pheromone ethyl oleate in honey bees

Honey bees allocate tasks along reproductive and non-reproductive lines: the queen mates and lays eggs, whereas the workers nurse the brood and forage for food. Among workers, tasks are distributed according to age: young workers nurse and old workers fly out and forage. This task distribution in the colony is further regulated by an increase in juvenile hormone III as workers age and by pheromones. One such compound is ethyl oleate (EO), a primer pheromone that delays the onset of foraging in young workers. EO is produced by foragers when they are exposed to ethanol (from fermented nectar) while gathering food. EO is perceived by younger bees via olfaction. We describe here the seasonal variation of EO production and the effects of Methoprene, a juvenile hormone analog. We found that honey bee workers biosynthesize more EO during the growing season than during the fall and winter months, reaching peak levels at late spring or summer. When caged workers were fed with syrup+d(6)-ethanol, labeled EO accumulated in the honey crop and large amounts exuded to the exoskeleton. Exuded levels were high for several hours after exposure to ethanol. Treatment with Methoprene increased the production of EO in worker bees, by speeding up its movement from biosynthetic sites to the exoskeleton, where EO evaporates. Crop fluid from bees collected monthly during the growing season showed a modest seasonal variation of in vitro EO biosynthetic activity that correlated with the dry and sunny periods during which bees could forage.

Sensory reception of the primer pheromone ethyl oleate

Social work force distribution in honeybee colonies critically depends on subtle adjustments of an age-related polyethism. Pheromones play a crucial role in adjusting physiological and behavioral maturation of nurse bees to foragers. In addition to primer effects of brood pheromone and queen mandibular pheromone—both were shown to influence onset of foraging—direct worker–worker interactions influence adult behavioral maturation. These interactions were narrowed down to the primer pheromone ethyl oleate, which is present at high concentrations in foragers, almost absent in young bees and was shown to delay the onset of foraging. Based on chemical analyses, physiological recordings from the antenna (electroantennograms) and the antennal lobe (calcium imaging), and behavioral assays (associative conditioning of the proboscis extension response), we present evidence that ethyl oleate is most abundant on the cuticle, received by olfactory receptors on the antenna, processed in glomeruli of the antennal lobe, and learned in olfactory centers of the brain. The results are highly suggestive that the primer pheromone ethyl oleate is transmitted and perceived between individuals via olfaction at close range.