The queen in relation to wax secretion and comb building in honeybees

Summary The role of the queen in relation to wax secretion and comb building in honeybees was analyzed with respect to queen status (mated, virgin and dead queens and queenlessness), and pheromones of the head and abdominal tergite of queens. Worker variables considered were colony size, percentage of bees bearing wax scales, wax scale weight, and weight of constructed combs.The amount of wax recovered from festoon bees and the percentage of festoon bees bearing wax were independent of queen status, the pheromones of queens and access to the queen. Colonies with full access to freely moving mated queens always constructed significantly more comb than those headed by virgin or dead queens as well as all permutations of caged and division board queens whose mandibular glands and/or abdominal tergite glands were operative or not.Despite pheromonal similarity of virgin queens to mated ones, colonies headed by virgin queens constructed as little comb as did queenless colonies. The bouquets of the mandibular glands did not differ significantly among queens nor was the amount of comb constructed correlated with pheromonal bouquet. Comb building is greatest among colonies having full access to freely moving queens but the stimulus for such building is not attributable to the 90DA, 9HDA and 10HDA components of the queen's mandibular gland secretions.     

Life expectancy and onset of foraging in the honeybee (Apis mellifera)

Predictions that precocious foraging in honeybee workers is a result of shortening of their life expectancy were tested in both laboratory and field experiments. In the laboratory experiment, we assessed the impact of anaesthesia with CO₂ and infection with Nosema apis on the lifespan of workers. In the field experiment, the age at onset of foraging was observed in groups of workers with different expected lifespans. In both the experiments, workers originating from one queen inseminated with the semen of one drone were divided into five groups. The first group was anaesthetized with CO₂ on the first day of life. Workers from the other three groups were individually inoculated with a constant number of N. apis spores on the 1st, 6th and 11th days of life. Workers from the fifth control group were neither treated with CO₂ nor inoculated in any way. Both the laboratory and field experiments revealed that anaesthetized and infected workers had shorter expected lifespans compared to control bees. Amongst infected workers, those inoculated earlier in life survived for a significantly shorter period of time in comparison to those infected later in life. In agreement with the expectation, the field experiment showed that anaesthetized and infected workers with shorter expected lifespan start foraging earlier than control workers. Amongst the infected workers, age at inoculation correlated with age at onset of foraging. This means that short-lived workers complete safe nest tasks and begin riskier foraging earlier in life. Our results provide a strong support for the hypothesis that the division of labour in eusocial insects is a consequence of the different expected worker lifespan and different risk associated with their tasks; however, they do not contradict other existing explanations.     

Second-order ocellar neurons in the brain of the honeybee (Apis mellifera)

Summary Electrophoretic injection of Procion Yellow M-R4 into the ocellar tract of the worker bee has revealed the following:Two types of giant axon run from the lateral ocellus to the circumesophageal neuropile, where one branches ipsilaterally and the other contralaterally. A third type comes from the median ocellus and can be traced into the cervical connectives. The largest dendritic complex is in the circumesophageal neuropile; in addition, fiber endings have been demonstrated in the following areas: in the subretinal region, along the optic commissure, in the medulla interna, in the subesophageal ganglion and between the neurosecretory cells of the pars intercerebralis. — The giant fibers are enclosed in a glial sheath.Three types of cell body are described. One is associated with the glia; another, larger cell type comprises giant-axon somata. The third type of cell is small, and cannot yet be identified.Some of the histological results are discussed with respect to the possible function of the ocellus.     

Hygropreference and brood care in the honeybee (Apis mellifera)

Terrestrial organisms need to limit evaporation from their bodies in order to maintain a homeostatic water balance. Owing to a large surface to volume ratio, arthropods are particularly susceptible to desiccation and have evolved behavioural and physiological mechanisms to conserve water. In social insects, water balance is also affected by the interactions between nestmates and by the architecture of the nest. For honeybees, humidity is particularly important for the brood because it affects the hatching success of eggs and because, unlike ants, honeybees cannot relocate their brood to parts of the nest with more favourable humidity. To advance the understanding of the water economy in honeybee nests, we investigated whether workers exhibit a hygropreference when exposed to a gradient of 24-90% relative humidity (RH) and whether the expression of this preference and their behaviour is affected by the presence of brood. The results show that young honeybee workers in the absence of brood exhibit a weak hygropreference for approximately 75% RH. When brood is present the expression of this preference is further weakened, suggesting that workers tend to the brood by distributing evenly in the gradient. In addition, fanning behaviour is shown to be triggered by an increase in humidity above the preferred level but not by a decrease. Our results suggest that humidity in honeybee colonies is actively controlled by workers.     

Inter-individual variation in nutrient balancing in the honeybee (Apis mellifera)

The Geometric Framework approach in nutritional ecology postulates that animals attempt to balance the consumption of different nutrients rather than simply maximizing energetic gain. The intake target with respect to each nutrient maximizes fitness in a specific dimension and any difference between individuals in intake target therefore represents alternative behavioral and fitness maximization strategies. Nutritional interactions are a central component of all social groups and any inter-individual variation in intake target should therefore have a significant influence on social dynamics. Using the honeybee colony as an experimental model, we quantified differences in the carbohydrate intake target of individual foragers using a capillary feeder (CAFE) assay. Our results show that the bees did not simply maximize their net energetic gain, but combined sugar and water in their diet in a way that brought them to an intake target equivalent to a 33% sucrose solution. Although the mean intake target with respect to the nutrients sucrose and water was the same under different food choice regimens, there was significant inter-individual variation in intake target and the manner in which individuals reached this target, a variation which suggests different levels of tolerance to nutrient imbalance. We discuss our results in the context of how colony performance may be influenced by the different nutrient balancing strategies of individual members and how such nutritional constraints could have contributed to the evolution of sociality.     

Ecdysteroid biosynthesis in workers of the European honeybee Apis mellifera L

We previously reported preferential expression of genes for ecdysteroid signaling in the mushroom bodies of honeybee workers, suggesting a role of ecdysteroid signaling in regulating honeybee behaviors. The organs that produce ecdysteroids in worker honeybees, however, remain unknown. We show here that the expression of neverland and Non-molting glossy/shroud, which are involved in early steps of ecdysteroid synthesis, was enhanced in the ovary, while the expression of CYP306A1 and CYP302A1, which are involved in later steps of ecdysone synthesis, was enhanced in the brain, and the expression of CYP314A1, which is involved in converting ecdysone into active 20-hydroxyecdysone (20E), was enhanced in the brain, fat body, and ovary. In in vitro organ culture, a significant amount of ecdysteroids was detected in the culture medium of the brain, fat body, and hypopharyngeal glands. The ecdysteroids detected in the culture medium of the fat body were identified as ecdysone and 20E. These findings suggest that, in worker honeybees, cholesterol is converted into intermediate ecdysteroids in the ovary, whereas ecdysone is synthesized and secreted mainly by the brain and converted into 20E in the brain and fat body.     

Pattern vision of the honeybee (Apis mellifera): the effect of pattern on the discrimination of location

This paper investigates how the pattern influences the discrimination of different locations of two or more areas of black, white or colour. The coloured patterns were made from two calibrated coloured papers that give contrast only to green receptors, or alternatively only to blue receptors. The patterns are fixed during training. It is found that the discrimination of translocation of two areas of colour involves green receptors and also blue receptors, and the resolution depends strongly on the pattern. Patterns that offer horizontal strips and up-down differences in locations are well resolved, even with no green contrast. Resolution of left-right reversal is greatly improved when the patterns promote fixation in the horizontal plane, as if green contrast is essential to stabilize the eye in yaw. The addition of radial bars with green contrast, a central black spot or a black surround, is particularly effective. The additions promote fixation, and would aid the detection of natural symmetrical objects. Accepted: 30 May 1999     

Engrailed expression and body segmentation in the honeybee Apis mellifera

Summary Honeybee embryos were stained with a monoclonal antibody raised against the Drosophila engrailed protein. The antibody was found to label rows of nuclei in the transverse grooves that form the earliest external sign of metameric germ band organization. These grooves demarcate metameric units about seven cell rows wide, of which about three rows with reduced apical cell surfaces account for the grooves. The en stripes appear in the grooves as soon as these form and grow from one to about four cells in width and thus completely overlap the groove. During the rudimentary germ band retraction, the grooves shift slightly backwards relative to both the en stripes and the trachdeal pits. The spatio-temporal pattern by which the series of grooves and stripes arises is quite striking. Both become visible first in the gnathal and thoracic regions, then in the pregnathal parts of the head and in the abdomen. The stripes arise essentially in an antero-posterior sequence. In addition, the earliest stripes to form display a pattern of alternating intensities whereas the later stripes, those in the abdomen, arise with approximately equal strength. The latter trait was earlier observed in the grasshopper, while the former is known from Drosophila where, however, the strong stripes correspond to the weak stripes in the honeybee.     

Visual discriminations of spokes, sectors, and circles by the honeybee (Apis mellifera)

A new cue for visual discrimination by the honeybee has been demonstrated. Bees detected the position of the centre of symmetry of radial patterns of spokes, sectors, and circles relative to their point of choice in the learning process, irrespective of the pattern. When trained with one of these patterns versus a blank target, the bees discriminated a shift in the position of the centre of symmetry by as little as 5 degrees , in some cases with unfamiliar test patterns. A pattern of spokes or rings also stabilized the vision of the bees in the horizontal plane so that the position of a plain black area could then be discriminated. In other experiments, bees discriminated half of a pattern of radial spokes or concentric circles from the other half, cut either vertically or horizontally, and irrespective of scale. Therefore these patterns were not detected by preformed combinations of orientation detectors or global templates with a single output. Instead, the crucial cue for detecting edges as radial or circular was the coincidence of responses of numerous local edge detectors having the appropriate convergence to a hub. Edges that converged towards a hub were detected by the bees as radial, and edges at right angles to these were parts of circles, irrespective of the actual pattern. Breaking the patterns of spokes or circles into rows of squares spoiled the discrimination if the squares were separately resolved. Alternatively, breaking the pattern into short bars that were separately resolved spoiled the discrimination when the bars subtended less than 3 degrees . The local feature detectors for spokes and circles therefore resembled those of the orientation detectors in being short, independent, and unable to span gaps of more than 3 degrees . In conclusion, radial and circular patterns were identified by the regional coincidences and convergence of local detectors of edge orientation, and the positions of the centres of symmetry were remembered as landmarks that helped locate the reward, but the patterns themselves were not remembered.     

ame-let-7调节蜜蜂劳动分工行为的研究进展

意大利蜜蜂是典型的社会性昆虫,存在与年龄相关的劳动分工行为,一直是人类研究动物行为的最佳模式生物。蜜蜂行为可塑性是劳动分工的重要特点,与其自身脑部神经结构变化、脑部基因表达变化及体内激素水平等有关。miRNAs与靶基因特异性结合,发挥多种生物调节功能。miR-let-7介导靶基因在动物发育及病理方面的研究较深,但对动物行为可塑性的调控机理尚未明确。本文就ame-let-7影响蜜蜂哺育蜂与采集蜂的劳动分工行为研究进行总结,主要从ame-let-7的表达模式、其对蜜蜂行为可塑性调节作用、对蜜蜂糖反应及学习记忆能力的影响及其相关靶基因的研究等方面进行归纳,为深入研究ame-let-7对蜜蜂行为可塑性的调控机制提供理论依据。     

Molecular cloning and characterization of a lipase from the honeybee Apis mellifera

Lipids perform essential and important functions, such as serving as an energy source for growth, development, reproduction, flight, starvation, and diapause in insects. Lipases are key enzymes involving in lipid metabolism and have been reported in several insect species. However, the molecular characterization of the pancreatic triacylglycerol lipase-like genes in honeybees has not been elucidated. Here, we describe the cDNA cloning and characterization of lipase from the honeybee Apis mellifera (Am-Lipase). Am-Lipase consists of 321 amino acids, including a Ser-Glu-His catalytic triad and a consensus active site motif GXSXG. Recombinant Am-Lipase protein degrades triglycerides, preferentially catalyzing the hydrolysis of long-chain fatty acids. Furthermore, the expression pattern of Am-Lipase seems to be a fat body-specific lipase, shows higher expression in forager bees, and is decreased under starvation conditions. Thus, our results suggest that Am-Lipase plays a role in the utilization of lipids stored in the fat body for lipid metabolism.     

Expression pattern of empty-spiracles,a conserved head-patterning gene,in honeybee (Apis mellifera) embryos

Empty-spiracle class homeodomain proteins have similar roles in anterior and head development in many animal species. We have identified a honeybee empty-spiracles gene and examined its expression in honeybee ovaries and embryos. The expression of honeybee empty-spiracles in embryos is similar to that reported for Drosophila and Tribolium, implying broad conservation of the role of this gene in insect embryogenesis. We also identify expression in somatic and germ-line cells of the ovary, not previously seen in other insect species.     

王浆主蛋白MRJP7基因在意大利蜜蜂体内的表达

王浆蛋白是蜂王浆生物功能的物质基础,是由王浆蛋白基因家族(mrjps)编码合成的。但部分家族成员如MRJP7在王浆中的含量极少甚至检测不到。基因功能与其在生物体内的时空表达特性相关,为探究mrjp7的生物学功能,本研究利用荧光定量PCR技术对mrjp7在不同发育时期的工蜂和成年工蜂、雄蜂和蜂王的不同组织部位的表达进行定量检测。结果显示mrjp7在成年雄蜂体内的表达水平最低,成年蜂王次之,且在它们的各不同组织部位之间的表达量差异较小。该基因在工蜂幼虫和蛹期的表达同样较低,但在羽化后9日龄前后的哺育蜂王浆腺和头部特异性高表达,这与哺育蜂分泌蜂王浆哺育幼虫和蜂王的功能是相适应的,该结果在转录水平上证实了mrjp7的营养功能,为进一步的研究和应用打下了理论基础。     

Antiapoptotic role of major royal jelly protein 8 of honeybee (Apis mellifera) venom

The dominant protein components of honeybee royal jelly (RJ) are major royal jelly proteins (MRJPs), which exhibit various biological properties. However, the biological basis of why bee venom contains MRJPs and what role MRJPs play in bee venom remains to be elucidated. This study reports the antiapoptotic role of MRJP 8 of Apis mellifera venom (AmMRJP 8) in melittin-treated mammalian cells. Recombinant AmMRJP 8 reduced caspase-3 activity and melittin-induced cell apoptosis. Additionally, recombinant AmMRJP 8 decreased the production levels of H₂O₂ and proinflammatory molecules. These results indicate that MRJP in bee venom plays a role in cell protection in bee venom-induced inflammatory responses.     

Antioxidant capacity of major royal jelly proteins of honeybee (Apis mellifera) royal jelly

Major royal jelly proteins (MRJPs) of honeybee royal jelly (RJ) exhibit antimicrobial and antioxidant activities. Although MRJPs of Apis mellifera RJ (AmMRJPs) responsible for antibacterial activity have been identified, AmMRJPs with antioxidant effects remain to be elucidated. Here we identified and compared the antioxidant activities of purified recombinant AmMRJPs 1–7, which are expressed in baculovirus-infected insect cells. Antioxidant assays of recombinant AmMRJPs 1–7 against H₂O₂ revealed that AmMRJPs reduce caspase-3 activity and oxidative stress-induced cell apoptosis and lead to increased cell viability. Consistent with these results, AmMRJPs 1–7 exhibit 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity and protect against oxidative DNA damage. These results indicate that AmMRJPs play a role as antioxidants in A. mellifera RJ.     

Antimicrobial activity of major royal jelly protein 8 and 9 of honeybee (Apis mellifera) venom

Honeybee venom is a complex mixture of toxic components, including major royal jelly protein (MRJP) 8 and 9. MRJP 8 and MRJP 9 are allergens, and MRJP 8 reduces melittin-induced cell apoptosis. However, their functional roles are poorly understood, and their antimicrobial activities have not been determined. In this study, the antimicrobial role of MRJP 8 and MRJP 9 of honeybee (Apis mellifera) venom (AmMRJP 8 and AmMRJP 9) was demonstrated. The presence of AmMRJP 8 and AmMRJP 9 in the secreted venom was observed using antibodies against recombinant AmMRJP 8 and AmMRJP 9 produced in baculovirus-infected insect cells. Recombinant AmMRJP 8 and AmMRJP 9 exhibited an inhibitory activity against microbial serine proteases. Consistent with their inhibitory activity, they induced structural damage by binding to microbial surfaces, resulting in a broad-spectrum antimicrobial activity against bacteria and fungi. They had little effect on hemolysis. Therefore, AmMRJP 8 and AmMRJP 9 could function as antimicrobial agents in honeybee venom.     

Early steps of cryopreservation of day one honeybee (Apis mellifera) embryos treated with low-frequency sonophoresis

Honeybees, major providers of pollination, are endangered in many areas. Embryo cryopreservation may be a very useful tool to maintain their genetic diversity. However, it is complex in insects, because embryos are chill sensitive and are surrounded by two protectant membranes, the chorion and vitelline. These membranes prevent penetration of cryoprotectant in the embryos. This study aimed to test different conditions of embryo preparation before cryopreservation, including low-frequency sonophoresis, a physical method of permeabilization, and passages through cryoprotectant solutions. Apis mellifera ligustica embryos were collected in artificial cell plugs 7.5 h after queens had been caged, in two different seasons (winter, spring) and were then incubated in vitro overnight (16.5 h). Embryos were individually sonicated and then incubated in three cryoprotectant baths (B1 = 10%, B2 = 20% and B3 = 40% of cryoprotectant) and quenched in liquid nitrogen. Artificial cell plugs and in vitro incubation device were efficient in producing future embryos hatching. Embryos stained ruby red with rhodamine B after sonophoresis treatment indicated that low-frequency ultrasound had permeabilized embryos. According to the treatment, different significant hatching rates were obtained after sonophoresis (up to 25%). After three cryoprotectant incubations, best hatching rates were obtained after 10 min in B1 and B2, and 40 s in B3. These results show that sonophoresis is an efficient tool to permeabilize the chorion and vitelline membrane of the day one honeybee embryo allowing a hatching rate of more than 20%. They also show that the season is an important variability factor.     

Tactile motor learning in the antennal system of the honeybee (Apis mellifera L.)

Honeybees fixed in small tubes scan an object within the range of the antennae by touching it briefly and frequently. In our experiments the animals were able to scan an object for several minutes with the antennae. After moving the object out of the range of the antennae, the animals showed antennal movements for several minutes that were correlated with the position of the removed object. These changes of antennal movements are called “behavioural plasticity” and are interpreted as a form of motor learning. Bees showed behavioural plasticity only for objects with relatively large surfaces. Plasticity was more pronounced in bees whose compound eyes were occluded. Behavioural plasticity was related to the duration of object presentation. Repeated presentations of the object increased the degree of plasticity. After presentation durations of 30 min the animals showed a significant increase of antennal positions related to the surface of the object and avoidance of areas corresponding to the edges. Behavioural plasticity was compared with reward-dependent learning by conditioning bees to objects. The results of motor learning and reward-dependent conditioning suggest that bees have tactile spatial memory. Accepted: 13 May 1997     

The preferences of the honeybee (Apis mellifera) for different visual cues during the learning process

By working with very simple images, a number of different visual cues used by the honeybee have been described over the past decades. In most of the work, the bees had no control over the choice of the images, and it was not clear whether they learned the rewarded pattern or the difference between two images. Preferences were known to exist when untrained bees selected one pattern from a variety of them, but because the preferences of the bees were ignored, it was not possible to understand how natural images displaying several cues were detected. The preferences were also essential to make a computer model of the visual system. Therefore experiments were devised to show the order of preference for the known cues in the training situation. Freely flying bees were trained to discriminate between a rewarded target with one pattern on the left side and a different one on the right, versus a white or neutral target. This arrangement gave the bees a choice of what to learn. Tests showed that in some cases they learned two or three cues simultaneously; in other cases the bees learned one, or they preferred to avoid the unrewarded target. By testing with different combinations of patterns, it was possible to put the cues into an order of preference. Of the known cues, loosely or tightly attached to eye coordinates, a black or blue spot was the most preferred, followed by strong modulation caused by edges, the orientation of parallel bars, six equally spaced spokes, a clean white target, and then a square cross and a ring. A patch of blue colour was preferred to yellow.