Photoperiod influences endogenous rhythm of ambient temperature selection by the honeybee Apis mellifera

The aim of this study was to evaluate whether the day–night cycle phase is a critical factor modulating diurnal rhythm of isolated honeybee's thermal preference or other factors are involved. The insects were exposed to standard (LD 12:12) and reversed (DL 12:12) photoperiods as well as to constant light and constant darkness conditions. Thermal preference and motor activity of honeybees were recorded for 3–5 days in a thermal gradient system. Under the standard (control) photoperiod conditions mean values of temperature selected by honeybees changed rhythmically within the period of about 24 h. Honeybees, exposed to the modified light–darkness cycle distinctly modified their rhythm of thermal preference. Under the reversed photoperiod conditions period of selected ambient temperature was much longer than before, until a complete reversal of the circadian oscillation was established at the end of the experiment. Experiments performed under constant light and constant darkness yielded undisturbed 24 h rhythms of both ambient temperature selection and locomotor activity. Under these conditions only a slight, nonsignificant flattening of the temperature selection curves was noticed. Both lack of substantial changes in the amplitude and occurring phase shifts of the rhythm, recorded in our experiments suggest its endogenous character. Our results prove that diurnal rhythm of ambient temperature selection by bee workers may be entrained by light–dark cycles. This implies a critical role of photoperiod in the modulation of nychthemeral oscillations of thermal preference in honeybees.     

Straight forward to the queen: pursuing honeybee drones (Apis mellifera L.) adjust their body axis to the direction of the queen

At a natural drone congregation area freeflying drones were attracted by a fast-moving queen dummy and the pursuits of drones were stereoscopically recorded (Fig. 1). The reconstruction of 192 flight paths from successfully approaching drones in chronological three dimensional sequences (Fig. 4) lead to the following results: 1. The alignment of the drone's longitudinal body axis coincides fairly well with the line connecting drone and queen (drone-queen-axis), its mean angular deviation from this line being only 14°. Angles between -5° and 5° occur most frequently (Fig. 5B). Thus, drones head straight to the queen. 2. Lateral deviations from the drone-queen-axis most frequently lie between — 30° and 30° (Fig. 5A) which corresponds to the drone's binocular visual field. 3. The drone's heading was continuously adjusted to the actual target, mean turning speed being 1890°/s. 4. The results lead to the conclusion that honeybee drones choose the shortest way to a fast and not predictably moving mate. A comparison with earlier observations suggests that a drone's mating success depends not only on his skills to win a race but also on his persistence within a group.     

The mating behaviour of the honeybee (Apis mellifera L.)

An account is given of the mating responses of free-flying drones towards a queen, and of the pheromones controlling these responses.     

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     

Differential antennal proteome comparison of adult honeybee drone, worker and queen (Apis mellifera L.)

To understand the olfactory mechanism of honeybee antennae in detecting specific volatile compounds in the atmosphere, antennal proteome differences of drone, worker and queen were compared using 2-DE, mass spectrometry and bioinformatics. Therefore, 107 proteins were altered their expressions in the antennae of drone, worker and queen bees. There were 54, 21 and 32 up-regulated proteins in the antennae of drone, worker and queen, respectively. Proteins upregulated in the drone antennae were involved in fatty acid metabolism, antioxidation, carbohydrate metabolism and energy production, protein folding and cytoskeleton. Proteins upregulated in the antennae of worker and queen bees were related to carbohydrate metabolism and energy production while molecular transporters were upregulated in the queen antennae. Our results explain the role played by the antennae of drone is to aid in perceiving the queen sexual pheromones, in the worker antennae to assist for food search and social communication and in the queen antennae to help pheromone communication with the worker and the drone during the mating flight. This first proteomic study significantly extends our understanding of honeybee olfactory activities and the possible mechanisms played by the antennae in response to various environmental, social, biological and biochemical signals.     

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.     

Comparative analysis of the intrinsic leg musculature of the American cockroach,Periplaneta americana (L.)

The American cockroach has a total of 368 muscles inserting on the post-coxal segments of its legs. By using a narrow morphological definition for delimiting individual muscles, it is shown (i) that the protrochanteral musculatures (23 muscles/leg) differ from the essentially identical meso- and metatrochanteral musculatures (24 and 26 muscles/leg) in number and disposition of extensors and in having a completely different flexor composition, and (ii) that the musculatures of the more distal segments of the legs are completely serially homologous, there being 2 muscles for moving each femur, 23 for each tibia, 7 for each first tarsomere, and 5 for each of the paired pretarsal claws. In all six legs, the trochanteral and tibial musculatures each contain single slender muscles that may be acting proprioceptively to measure the angular displacements between, respectively, the coxas and trochanters, and the femurs and tibias. Neurological and phylogenetic considerations are used to demonstrate why a narrow morphological definition should be employed, and why the widely used functional definition of Snodgrass ('35) is not only fallacious on evolutionary grounds, but also leads to making erroneous conclusions regarding the manner in which insect musculature is controlled by the insect central nervous system. Finally, it is hypothesized that the physiological limitations imposed by having an open circulatory system and the problems inherent in the neural control of large muscles may have been major evolutionary factors in forcing insects to use many slender muscles to control their body movements.     

Associative learning and discrimination of motion cues in the harnessed honeybee Apis mellifera L.

We previously studied a conditioning paradigm to associate the proboscis extension reflex (PER) with monochromatic light (conditioned stimulus; CS) in harnessed honeybees. Here, we established a novel conditioning paradigm to associate the PER with a motion cue generated using graphics interchange format (GIF) animations with a speed of 12 mm/s speed and a frame rate of 25 Hz as the CS, which were projected onto a screen consisting of a translucent circular cone that largely covered the visual field of the harnessed bee using two liquid crystal projectors. The acquisition rate reached a plateau at approximately 40% after seven trials, indicating that the bees were successfully conditioned with the motion cue. We demonstrated four properties of the conditioning paradigm. First, the acquisition rate was enhanced by antennae deprivation, suggesting that sensory input from the antennae interferes with the visual associative learning. Second, bees conditioned with a backward-direction motion cue did not respond to the forward-direction, suggesting that bees can discriminate the two directions in this paradigm. Third, the bees can retain memory for motion cue direction for 48 h. Finally, the acquisition rate did not differ significantly between foragers and nurse bees. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tyrosine hydroxylase in the cerebral ganglia of the American cockroach (Periplaneta americana L.): an immunohistochemical study

We have investigated the distribution of tyrosine-hydroxylase-like immunoreactivity in the cerebral ganglia of the American cockroach, Periplaneta americana. Groups of tyrosine-hydroxylase-immunoreactive cell bodies occur in various parts of the three regions of the cerebral ganglia. In the protocerebrum, single large neurons or small groups of neurons are located in the lateral neuropil, adjacent to the calyces, and in the dorsal portion of the pars intercerebralis. Small scattered cell bodies are found in the outer layers of the optic lobe, and clusters of larger cell bodies can be found in the deutocerebrum, medial and lateral to the antennal glomeruli. Thick bundles of tyrosine-hydroxylase-positive nerve fibers traverse the neuropil in the proto- and deutocerebrum and innervate the glomerular and the nonglomerular neuropil with fine varicose terminals. Dense terminal patterns are present in the medulla and lobula of the optic lobe, the pars intercerebralis, the medial tritocerebrum, and the area surrounding the antennal glomeruli, the central body and the mushroom bodies. The pattern of tyrosine-hydroxylase-like immunoreactivity is similar to that previously described for catecholaminergic neurons, but it is distinctly different from the distribution of histaminergic and serotonergic neurons.     

Genetic variance of mating frequency in the honeybee (Apis mellifera L.)

Summary. The genetic variance of queen mating frequency was studied in honeybees (Apis mellifera carnica). Worker offspring (N = 966) of 28 naturally mated half sister-queens (r = 0.25) from seven unrelated breeding lines were genotyped at four DNA microsatellites. The mating frequencies of the queens were derived from the offspring genotypes. The number of observed matings per queen ranged from 10 to 28 with an average of 17.32 ± 1.10 (number of estimated matings: 24.94 ± 2.51; number of effective matings: 20.09 ± 1.73). Half-sib analyses of the breeding lines were used to estimate heritability. Heritability was h² = 0.449 ± 0.135 for the estimated number of matings and h² = 0.262 ± 0.103 for the number of effective males, which are both significantly different from zero. We conclude that a high genetic variance for polyandry in honeybees can be favored by balanced selection between individual queen and colony level.Received 16 October 2003; revised 4 May 2004; accepted 4 May 2004.     

Serotonin-immunoreactive visceral nerves and neurohemal system in the cockroach Periplaneta americana (L.)

Summary By use of the indirect immunofluorescent technique applied to whole mounts of tissues and organs of the American cockroach, Periplaneta americana, serotonin-like reactivity has been demonstrated in an extensive meshwork of fibers on the surface of nerves of the subesophageal ganglion and the tritocerebrum. This meshwork appears to serve the neurohemal release of serotonin. In addition, several of these nerves contain two or more centrally located axons that appear to be serotonergic. The corpora cardica show immunoreactivity but do not appear to be a principal release site for serotonin. The nerves and ganglia of the stomadeal nervous system show immunoreactivity for serotonin and provide serotonin-positive innervation to the salivary glands and the visceral muscles of the stomodeum and midgut. The heart, hindgut, Malpighian tubules, fat body, and skeletal muscles all appear to lack serotonin immunoreactivity.     

Estimating the genetic variance of group characters: social behaviour of honeybees (Apis mellifera L.)

Summary A new approach is presented to estimate the genetic variance of social behaviour of groups. Honeybees (Apis mellifera L.) are used as an example for highly social organisms. Most characters of economic importance strongly rely on collective group characters of honeybee colonies. The average relatedness between small groups of workers of one honeybee colony can be estimated using a discrete multinomial distribution. The genetic variance of a social behaviour (alarm behaviour) of groups of honeybee workers is estimated with the intraclass correlation between groups within a colony. In two populations tested, the coefficient of genetic determination was high (0.96–0.98) indicating that the metabolic bio-assay used was only weakly affected by environmental effects.     

Venereal and vertical transmission of deformed wing virus in honeybees (Apis mellifera L.)

Deformed wing virus (DWV) infected semen was used for artificial insemination of DWV-free virgin queens. High titres of DWV could subsequently be detected not only in the spermatheca, but also in the ovaries, demonstrating venereal transmission of DWV in honey bees. Subsequent vertical transmission of the virus to the progeny of DWV infected queens was also demonstrated. Neither transmission route is 100% effective. Whether venereal transmission of DWV occurs during natural mating remains to be determined. The implications for the use, sale and transport of semen samples for artificial insemination are discussed.     

Laying workers in queenless honeybee (Apis mellifera L.) colonies have physiological states similar to that of nurse bees but opposite that of foragers

Honeybee workers shift their labors from nursing their brood to foraging according to their age after eclosion. When the queen is lost from the colony, however, some workers become 'laying workers' whose ovaries develop to lay eggs. Here we investigated whether the physiological state of laying workers is more similar to that of nurse bees or foragers by examining the hypopharyngeal gland (HPG) and hemolymph vitellogenin titers. In a normal colony, nurse bees have well-developed HPGs that synthesize 'major royal jelly proteins' and high hemolymph vitellogenin titers, whereas foragers have shrunken HPGs that synthesize 70-kDa alpha-glucosidase and low hemolymph vitellogenin titers. In queenless colonies, however, workers with developed ovaries (laying workers) tended to have more developed HPGs and to synthesize major royal jelly proteins, whereas workers with shrunken HPGs tended to synthesize alpha-glucosidase and to have undeveloped ovaries. Furthermore, the workers with developed ovaries had higher vitellogenin titers than nurse bees, whereas those with undeveloped ovaries had lower vitellogenin titers. These findings indicate that the physiological state of laying workers is similar to that of nurse bees, but opposite that of foragers.     

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