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.     

The inhibiting effect of the queen bee (Apis mellifera L.) foot-print pheromone on the construction of swarming queen cups

Experiments were conducted to determine the rôle of population density of queenright honey bee colonies, and that of the queen bee pheromonal secretions, on the induction and inhibition of swarming queen cup construction during swarming and non-swarming seasons. Construction of queen cups was induced experimentally in overcrowded queenright colonies, during winter, which is a non-swarming season. This construction was induced by high population density of bee workers: above a threshold of 2.3 bee workers/ml there was a relationship between the number of cups constructed and the colony density. During the swarming season a relationship was established between the free volume of a hive (population density) and the number of queen cups constructed: 1.5 cups in a colony that occupied 80,960 ml, compared with 77 cups in a colony hived within a volume of 20, 240 ml. Observations of the queen's movements upon combs in colonies of high and normal population densities showed that in an overcrowded colony the queen bee was almost absent from the bottom edges of the comb, where queen swarming cups and cells are constructed. The tarsal glands of queens are located in the fifth tarsomere and the glandular oily secretion is deposited by the foot-pads upon the combs surface. The rate of secretion by the queen's tarsal glands was about 13 times higher than by those of the workers. A bioassay for testing the inhibitory effects of the queen's glandular extracts on the construction of queen cups was developed. It was based on increasing worker bee population densities, and can be used effectively throughout the year in a subtropical climate.The application of tarsal and mandibular glands' secretion to comb bottom edges in overcrowded colonies (bioassay) caused the inhibition of queen cup construction. None of these two secretions affected construction of these cups when applied separately. We presume that due to colony overcrowding the queen bee is unable to deposit the non-volatile secretions from tarsal glands along the comb edges and that the deficiency of the foot-print pheromone triggers the construction of swarming cups along the non-inhibited areas.     

Effect of primer pheromones and pollen diet on the food producing glands of worker honey bees (Apis mellifera L.)

Cooperative brood care is highly developed in the honey bee such that workers called nurses use their hypopharyngeal and mandibular glands to biosynthesize proteinaceous secretions that are progressively provisioned to larvae. The role that honey bee primer pheromones play in the functional physiology of food producing glands was examined. The combined and separate effects of queen mandibular pheromone (QMP) and brood pheromone (BP) on amount of protein extractable from hypopharyngeal and mandibular glands of workers reared for 12 days with and without pollen diets was measured. In rearing environments with a pollen diet, BP, and QMP + BP pheromone treatments significantly increased extractable protein from both glands. Bees reared with QMP + pollen had amounts of protein extractable from both glands that were not significantly different from control bees (no pheromones, no pollen). Pollen in the diet alone significantly increased amounts of protein extractable from glands versus control. In rearing environments without pollen, QMP + BP had a synergizing effect on amount of protein in both glands. The QMP + BP treatment was the only rearing environment without a pollen diet where protein amounts were significantly greater than the control. The synergizing effect of QMP + BP on extractable mandibular and hypopharyngeal gland protein suggests a highly derived role for the combined effect of these two primer pheromones on honey bee cooperative brood care. Mandibular gland area was significantly and positively correlated with extractable protein. Amounts of extractable protein from both glands declined significantly with age of workers in all treatments. However, treatment significantly affected rate of decline. The adaptive significance of gland protein amounts in response to pheromones and pollen diet are discussed.     

Influence of temperature and carbon dioxide concentration on juvenile hormone titre and dependent parameters of adult worker honey bees (Apis mellifera L.)

It is known that juvenile hormone plays an important role in the regulation of labour division and of the different life spans, and that the microclimate of the bee hive is characterized by its high CO₂ concentration and its varying temperature depending on the presence of brood.We have investigated the influence of microclimates characteristic of breeding and broodless areas on the juvenile hormone titre in the haemolymph and whole body extracts, on the corpora allata in vitro activity, on the degradation of juvenile hormone and on the dry weight of the hypopharyngeal glands using bees of known ages. A microclimate of 35°C and 1.5% CO₂, as observed in the breeding area, induces a rapid and pronounced increase in the juvenile hormone titre. On the other hand, this titre remains low in bees kept at 27°C and 1.5% CO₂, a microclimate associated with broodless combs. Rates of juvenile hormone synthesis by corpora allata in vitro were found to be extremely low, even in the presence of farnesenic acid, and not related to the juvenile hormone titre. In vitro incubation of juvenile hormone in haemolymph revealed no degradation while injected juvenile hormone was found to be degraded and taken up by the gut at rates only weakly correlated with the juvenile hormone titre.We propose a hypothetical model for the regulation of the juvenile hormone titre as well as the course of labour division by the varying microclimates observed in the bee hive.     

Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios

This is the first study where the systematic application of theories and techniques used in mammalian sperm cryopreservation have been applied to honey bee (Apis mellifera L.) semen as a means to improve postthaw viability of cryopreserved sperm. Six newly designed diluents, three cryoprotectants (dimethyl sulfoxide, DMA, glycerol), and five diluent:semen ratios (1:1, 3:1, 6:1, 9:1, and 12:1) were tested. In addition, the sperm freezing tolerance of three honey bee strains was evaluated. Specific protocols were designed to control semen freezing and thawing rates. Sperm motility was assessed visually, whereas sperm viability was assessed using SYBR-14 and propidium iodide fluorescent stains. Diluent treatments did not affect fresh (nonfrozen) sperm viability yet affected fresh sperm motility (P < 0.05). Based on these assessments, two diluents were chosen and used in all successive cryopreservation experiments. Using the selected diluents, semen was collected at various diluent:semen ratios, along with one of the three cryoprotectants. Semen collected at high dilution ratios, using a hypotonic antioxidant diluent containing catalase, in combination with dimethyl sulfoxide, provided higher postthaw sperm viability than that of all other combinations tested (68.3 ± 5.4%; P < 0.05). Using this combination of dilution ratio, diluent, and cryoprotectant, there were no differences among honey bee strains for postthaw sperm viability (P = 0.805). Nevertheless, these new semen dilution and freezing methods improved postthaw viability of sperm to levels that could theoretically sustain worker populations in colonies, thus providing potential for further optimization of cryopreservation techniques for the genetic preservation and improvement of honey bee genotypes.     

Functional analysis of the honeybee (Apis mellifera L.) salivary system using proteomics

In insects, specific proteins and physiologically active molecules whose functions are related to their lifestyles are secreted from the salivary system. To investigate proteins/molecules related to the sociality of the European honeybee (Apis mellifera L.), we performed a proteomic analysis of the honeybee salivary system. The honeybee salivary system comprises two secretory glands: the postcerebral gland (PcG) and the thoracic gland (TG), both of which are connected to a common duct that opens in the mouthpart. Although most (31 out of 35) of the major proteins identified from the PcG and TG were housekeeping proteins, the spot intensities for aldolase and acetyl-CoA acyltransferase 2 were stronger in the PcG than in the TG in the 2-dimensional gel electrophoresis. Immunoblotting confirmed that the expression of these proteins was stronger in the PcG than in the TG, whereas expression was almost not detectable in the hypopharyngeal gland (HpG), suggesting that carbohydrate metabolism is enhanced in the honeybee PcG. In addition, imaginal disc growth factor 4 (IDGF4) was synthesized in the honeybee salivary system. Immunoblotting indicated IDGF4 expression was very strong in the PcG, moderate in the TG, and very weak in the HpG. A considerable amount of IDGF4 was detected in the royal jelly, while less was detected in honey, strongly suggesting that the honeybee salivary system secretes IDGF4 into the royal jelly and honey. The secreted IDGF4 might therefore affect growth and physiology of the other colony members.     

Pheromonal activity and fine structure of the mandibular glands of honeybee drones (Apis mellifera L.) (Insecta,Hymenoptera, Apidae)

Experiments were conducted to determine the role of drone mandibular gland secretions in attracting flying drones and the effect of age on the secretory activity. Extracts of mandibular glands and of cephalic tissues were applied to cotton lures which were attached below balloons tethered at 8–12 m above the ground. Most flying drones were attracted to extracts of mandibular glands but a few were drawn to other cephalic tissues or to solvent controls. Histological and electron microscope studies showed that the structure of the tiny (0.12 mm long) mandibular gland varied according to age. Its secretory activity in 0–3-day old drones was evident from the abundant rough endoplasmic reticulum. At 7-days the glands were fully developed. After 9 days the glands were no longer active and showed an autolytic process; the product was stored in the gland lumen for further emission during drone mating flights.     

Insights into the dynamics of hind leg development in honey bee (Apis mellifera L.) queen and worker larvae - A morphology/differential gene expression analysis

Phenotypic plasticity is a hallmark of the caste systems of social insects, expressed in their life history and morphological traits. These are best studied in bees. In their co-evolution with angiosperm plants, the females of corbiculate bees have acquired a specialized structure on their hind legs for collecting pollen. In the highly eusocial bees (Apini and Meliponini), this structure is however only present in workers and absent in queens. By means of histological sections and cell proliferation analysis we followed the developmental dynamics of the hind legs of queens and workers in the fourth and fifth larval instars. In parallel, we generated subtractive cDNA libraries for hind leg discs of queen and worker larvae by means of a Representational Difference Analysis (RDA). From the total of 135 unique sequences we selected 19 for RT-qPCR analysis, where six of these were confirmed as differing significantly in their expression between the two castes in the larval spinning stage. The development of complex structures such as the bees’ hind legs, requires diverse patterning mechanisms and signaling modules, as indicated by the set of differentially expressed genes related with cell adhesion and signaling pathways.     

The spermathecal complex in Ips typographus (L.): Differentiation of the spermathecal gland related to age and reproductive state

The spermathecal complex of the bark beetle, Ips typographus, comprises the following elements: spermathecal duct, spermatheca and spermathecal gland. The spermathecal duct connects the vagina and the spermatheca and consists of a cuticular tube surrounded by an epithelial layer and circular muscles. The spermatheca is bottle-shaped and has a cuticle-lined lumen. Muscles are attached to both ends of the spermatheca. The spermathecal gland which is connected to the spermatheca possesses three cell types: glandular, hypodermal, and ductule. The glandular cells have different structural characteristics depending on the age and reproductive state of the females. After the emergence of the brood, two different kinds of secretory material are present in the glandular cells. There is evidence that one type of secretion is emitted during the first few days after brood emergence, while the other type accumulates to be secreted during later stages.