Jakob Wegener

Regulation of hypopharyngeal gland activity and oogenesis in honey bee (Apis mellifera) workers.

In the honey bee, vitellogenin has several functions in addition to egg provisioning. Among others, it serves as a precursor to brood food proteins secreted by the hypopharyngeal glands of worker bees. In queenless workers with developing gonads, oogenesis and development of the hypopharyngeal glands are correlated. Here we describe two experiments that explored whether this relationship also exists in non-reproductive workers, and investigated a possible role of ecdysteroid hormones in the regulation of vitellogenin uptake. In the first experiment, the correlation between oocyte length and hypopharyngeal gland development was measured in workers before and after de-queening. In the second experiment, we induced middle-aged bees with resting glands to suddenly initiate brood care behaviour, and measured haemolymph ecdysteroid and vitellogenin titres. A strong positive relationship existed between morphometrical parameters of hypopharyngeal glands and ovaries in both queenless and queenright (functionally sterile) workers. No response of ecdysteroid titres to the addition of brood was detected in experiment 2, but high concentrations were measured in a small group of bees characterised by the possession of oocytes on the brink of yolk incorporation. We conclude that hypopharyngeal glands may belong to a previously described group of reproduction-related traits that are pleiotropically regulated in workers. A possible role for ecdysteroids in honey bee reproduction is discussed.

Physiological consequences of prolonged nursing in the honey bee.

Abstract.Honey bee workers normally produce brood food at an age of 5 to 15 days. However, natural events like swarming or brood diseases may lead to the occurrence of over-aged nurses. Here we investigated the physiological consequences of prolonged nursing for both the nurses and the brood they rear, and tried to separate the effects of chronological age and of task affiliation on some important physiological parameters. Brood was reared in groups of colonies with either a normal age structure or with moderately over-aged workers.The haemolymph concentrations of total protein and vitellogenin, the development of mandibular and hypopharyngeal glands, and the activity of α-glucosidase in the hypopharyngeal glands of nurses from these groups of colonies were compared. Moreover, we used the fertility of young workers reared by normal- and overaged nurses as a bioindicator for the quality of the brood care they had received. It showed that parameters linked to the production of brood food proteins remained stable in over-aged nurses, whereas the development of mandibular glands regressed. Workers reared by over-aged nurses had more ovarioles and showed stronger ovary development under queenless conditions. Our results indicate that while over-aged nurses remain capable of producing brood food, they are not functionally equivalent to young nurses. The partial degeneration of the mandibular glands normally occurring at the end of the nursing period cannot be prevented by prolonged nursing. The distinct phenotype of workers reared by old nurses raises the question of possible age-related specialisations among nurses in colonies with a normal age structure.

Conservation of honey bee (Apis mellifera) sperm phospholipids during storage in the bee queen — A TLC/MALDI–TOF MS study

The honey bee (Apis mellifera) is characterized by a high degree of phenotypic plasticity of senescence-related processes, and has therefore become a model organism of gerontological research. Sperm of honey bee drones can remain fertile for several years within the storage organ of queens. The reason for this longevity is unknown, but the suppression of lipid peroxidation seems to play a decisive role. Here, we examined the questions of whether spermatheca- and in vitro-stored honey bee sperm are indeed resistant to lipid peroxidation, and whether the nature of sperm lipids could explain this resistance. The lipid composition of bee sperm was determined by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) combined with thin-layer chromatography (TLC). The positive ion mass spectra of drone sperm lipids are dominated by two glycerophosphocholine (GPC) species, although small amounts of sphingomyelins (SM) and glycerophosphoethanolamines (GPE) are also detectable after TLC. Alkyl/acyl and alkenyl/acyl compounds of GPC, and alkyl/acyl as well as diacyl compounds of GPE were detected containing oleyl, oleoyl, palmityl and palmitoyl as the most abundant residues. Assignments of all compounds have been additionally verified by enzymatic digestion and exposition to HCl. During incubation of sperm in the presence of air, characteristic lipid oxidation products such as lysophosphatidylcholine (LPC) appear. Inside the spermatheca, however, sperm lipids are obviously protected from oxidation and their composition does not change, even if they are stored over years. Our data support the view that the membrane composition of honey bee sperm could help to explain the extraordinary longevity of these cells.

New insights into the roles of juvenile hormone and ecdysteroids in honey bee reproduction

In workers of the Western honeybee, Apis mellifera, juvenile hormone (JH) and ecdysteroids regulate many aspects of age polyphenism. Here we investigated whether these derived functions in workers have developed by an uncoupling of endocrine mechanisms in adult queens and workers, or whether parallels can be found between the roles of the two hormones in both castes. We looked at yolk protein metabolism as a process central to the physiology of both queens and workers, and at sperm storage as a feature of the queen alone. Queens of differing fertility status (virgin, virgin but CO2-treated, inseminated, freshly laying and 1-2 years-old) were compared regarding vitellogenin (Vg), JH and ecdysteroid-titers in their hemolymph, as well as ovarian yolk protein and spermathecal gland composition. Our results showed that hormone titres were unrelated to the composition of spermathecal glands. JH-concentrations in the hemolymph were low in the groups of queens characterized by yolk uptake into the ovaries, and high in pre-vitellogenic queens or animals that were forced to interrupt egg-laying by caging. Ecdysteroid-concentrations were higher in untreated virgins than after insemination or during egg-laying. They were not affected by the caging of queens. These patterns of hormone changes were parallel to those known from worker bees. Together, these findings suggest a conserved role for JH as repressor of vitellogenin uptake into tissues, and for ecdysteroids in preparing tissues for this process. An involvement of the two hormones in the regulation of sperm storage seems unlikely. Our results add to the view that JH and ecdysteroids act similarly on the yolk protein metabolism of both castes of A. mellifera. This may imply that it was the biochemical versatility of Vg rather than that of hormonal regulatory circuits that allowed for the functional separation of the two castes.

In vivo validation of in vitro quality tests for cryopreserved honey bee semen.

Development of cryopreservation protocols for honey bee semen is hampered by the lack of validated laboratory tests that allow the prediction of in vivo performance of frozen-thawed semen. Here we analyzed correlations between seven in vitro tests and indicators of semen performance after insemination. These tests included measures of motility, cell conformation, and membrane permeability before and after exposure to physiochemical stress. We show that the proposed protocol for motility measurement yields results that correlate well with the number of sperm reaching the storage organ of queens (correlation coefficient ρ=0.67) and the proportion of viable eggs in inseminated queens (ρ=0.48). The conventional live/dead assay of membrane permeability by dual fluorescent staining and a new test based on the leakage of the glycolytic enzyme glucose-phosphate-isomerase (GPI) from damaged cells were also correlated to the number of sperm reaching the spermatheca (ρ=0.54 and -0.61, respectively). We conclude that motility, live/dead-staining and the assay for GPI-leakage are valuable tools for the improvement of cryopreservation of honey bee semen.

Proteome Analysis of the Hemolymph, Mushroom Body, and Antenna Provides Novel Insight into Honeybee Resistance against Varroa Infestation

Varroa destructor has been identified as a major culprit responsible for the losses of millions of honeybee colonies. Varroa sensitive hygiene (VSH) is a suite of behaviors from adult bees to suppress mite reproduction by uncapping and/or removing mite infested pupae from a sealed brood. Despite the efforts to elucidate the molecular underpinnings of VSH, they remain largely unknown. We investigated the proteome of mushroom bodies (MBs) and antennae of adult bees with and without VSH from a stock selected for VSH based on their response to artificially Varroa-infected brood cells by near-infrared camera observation. The pupal hemolymph proteome was also compared between the VSH-line and the line that was not selected for VSH. The identified 8609 proteins in the hemolymph, MBs, and antennae represent the most depth coverage of the honeybee proteome (>55%) to date. In the hemolymph, the VSH-line adapts a unique strategy to boost the social immunity and drive pupal organogenesis by enhancing energy metabolism and protein biosynthesis. In MBs, the up-regulated proteins implicated in neuronal sensitivity suggest their roles to promote the execution of VSH by activation of synaptic vesicles and calcium channel activities. In antennae, the highly expressed proteins associated with sensitivity of olfactory senses and signal transmissions signify their roles by inputting a strong signal to the MBs for initiating VSH. These observations illustrate that the enhanced social immunities and olfactory and neuronal sensitivity play key roles in the combat against Varroa infestation. The identified candidate markers may be useful for accelerating marker-associated selection for VSH to aid in resistance to a parasite responsible for decline in honeybee health.

Suppression of worker fertility in the honey bee (Apis mellifera) by treatment with X-rays

Summary Breeding of the honey bee normally involves measurement of traits at the colony level. Nevertheless, there are traits that can be observed at the level of the individual worker. In this case, workers with interesting features are artificially induced to partheno-genetically produce offspring. In order to promote fertility in the worker bee of interest, it is useful to suppress the fertility of all other workers. In this study, our aim was to produce sterile bees by the application of ultra-hard X-rays. We irradiated capped brood of different ages with a dose of 20 Gy. This treatment suppressed fertility in all treated workers, while controls laid normal amounts of eggs. However, rare cases of fully developed ovaries occurred in workers when pupae were irradiated 0–4 days prior to emergence. In these bees, ovary development was retarded compared to controls. Hypopharyngeal glands degenerated more quickly in irradiated bees than in the controls, and mortality was heightened. Production of wax also was reduced. Radiosensibility of pupae dropped after approximately 110 h of age, egg and larval stages included. Given the rapid degeneration of hypopharyngeal glands in the irradiated bees, the usefulness of irradiated workers for breeding from single workers seems doubtful.

Secondary biomarkers of insecticide-induced stress of honey bee colonies and their relevance for overwintering strength.

The evaluation of pesticide side-effects on honeybees is hampered by a lack of colony-level bioassays that not only are sensitive to physiological changes, but also allow predictions about the consequences of exposure for longer-term colony productivity and survival. Here we measured 28 biometrical, biochemical and behavioural indicators in a field study with 63 colonies and 3 apiaries. Colonies were stressed in early summer by feeding them for five days with either the carbamate growth regulator fenoxycarb or the neurotoxic neonicotinoid imidacloprid, or left untreated. Candidate stress indicators were measured 8-64 days later. We determined which of the indicators were influenced by the treatments, and which could be used as predictors in regression analyses of overwintering strength. Among the indicators influenced by fenoxycarb were the amount of brood in colonies as well as the learning performance and 24h-memory of bees, and the concentration of the brood food component 10HDA in head extracts. Imidacloprid significantly affected honey production, total number of bees and activity of the immune-related enzyme phenoloxidase in forager bee extracts. Indicators predictive of overwintering strength but unrelated to insecticide feeding included vitellogenin titer and glucose oxidase-activity in haemolymph/whole body-extracts of hive bees. Apart from variables that were themselves components of colony strength (numbers of bees/brood cells), the only indicator that was both influenced by an insecticide and predictive of overwintering strength was the concentration of 10HDA in worker bee heads. Our results show that physiological and biochemical bioassays can be used to study effects of insecticides at the colony level and assess the vitality of bee colonies. At the same time, most bioassays evaluated here appear of limited use for predicting pesticide effects on colony overwintering strength, because those that were sensitive to the insecticides were not identical with those that were predictive of colony overwintering. Our study therefore illustrates the difficulties involved in evaluating the economic/ecological significance of pesticide-induced stress in honey bee field studies.

Pathogenesis of varroosis at the level of the honey bee (Apis mellifera) colony

The parasitic mite Varroa destructor, in interaction with different viruses, is the main cause of honey bee colony mortality in most parts of the world. Here we studied how effects of individual-level parasitization are reflected by the bee colony as a whole. We measured disease progression in an apiary of 24 hives with differing degree of mite infestation, and investigated its relationship to 28 biometrical, physiological and biochemical indicators. In early summer, when the most heavily infested colonies already showed reduced growth, an elevated ratio of brood to bees, as well as a strong presence of phenoloxidase/prophenoloxidase in hive bees were found to be predictors of the time of colony collapse. One month later, the learning performance of worker bees as well as the activity of glucose oxidase measured from head extracts were significantly linked to the timing of colony collapse. Colonies at the brink of collapse were characterized by reduced weight of winter bees and a strong increase in their relative body water content. Our data confirm the importance of the immune system, known from studies of individually-infested bees, for the pathogenesis of varroosis at colony level. However, they also show that single-bee effects cannot always be extrapolated to the colony as a whole. This fact, together with the prominent role of colony-level factors like the ratio between brood and bees for disease progression, stress the importance of the superorganismal dimension of Varroa research.

New Methods and Media for the Centrifugation of Honey Bee (Hymenoptera: Apidae) Drone Semen

ABSTRACT Centrifugation of Apis mellifera L. drone semen is a necessary step in the homogenization of semen pools for the enlargement of the effective breeding population, as well as in the collection of semen by the so-called washing technique. It is also of interest for the removal of cryoprotectants after cryopreservation. The adoption of methods involving semen centrifugation has been hampered by their damaging effect to sperm. Here, we tested four new diluents as well as three additives (catalase, hen egg yolk, and a protease inhibitor), using sperm motility and dual fluorescent staining as indicators of semen quality. Three of the new diluents significantly reduced motility losses after centrifugation, as compared with the literature standard. Values of motility and propidium iodide negativity obtained with two of these diluents were not different from those measured with untreated semen. The least damaging diluent, a citrate-HEPES buffer containing trehalose, was then tested in an insemination experiment with centrifuged semen. Most queens receiving this semen produced normal brood, and the number of sperm reaching the storage organ of the queen was not significantly different from that in queens receiving untreated semen. These results could improve the acceptance of techniques involving the centrifugation of drone semen. The diluent used in the insemination experiment could also serve as semen extender for applications not involving centrifugation.