Detection of coloured stimuli by honeybees: minimum visual angles and receptor specific contrasts

Honeybees Apis mellifera were trained to distinguish between the presence and the absence of a rewarded coloured spot, presented on a vertical, achromatic plane in a Y-maze. They were subsequently tested with different subtended visual angles of that spot, generated by different disk diameters and different distances from the decision point in the device. Bees were trained easily to detect bee-chromatic colours, but not an achromatic one. Chromatic contrast was not the only parameter allowing learning and, therefore, detection: _min, the subtended visual angle at which the bees detect a given stimulus with a probability P ₀ = 0.6, was 5° for stimuli presenting both chromatic contrast and contrast for the green photoreceptors [i.e. excitation difference in the green photoreceptors, between target and background (green contrast)], and 15° for stimuli presenting chromatic but no green contrast. Our results suggest that green contrast can be utilized for target detection if target recognition has been established by means of the colour vision system. The green-contrast signal would be used as a far-distance signal for flower detection. This signal would always be detected before chromatic contrast during an approach flight and would be learned in compound with chromatic contrast, in a facilitation-like process.     

The fine structure of the tarsal glands of the honeybee Apis mellifera L. (Hymenoptera)

Summary Tarsal glands are located in the 6th tarsomere of adult honeybee queens, workers and drones. Their structural features are not cast or sex specific. The glandular epithelium is lined by a thin endocuticular layer. A cuticular pocket is formed from a postimaginal delamination of the cuticle secreted by the glandular epithelium. The apical plasma membrane of the glandular cells shows numerous cristae and microvilli lining large crypts that communicate with the subcuticular space. Pinocytotic vesicles, multivesicular bodies and residual dense bodies are present in the apical part of the glandular cells. The RER is well developed in perinuclear and basal parts of the glandular cells, but the Golgi apparatus is a discrete organelle without secretory granules. No exocytotic secretory structures were observed. To reach the glandular pocket, the non-proteinaceous secretory product must pass across the subcuticular space, the cuticular intima, the space between the intima and the cuticular wall, and the cuticular wall of the glandular pocket.     

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.     

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.     

Interadult feeding of jelly in honeybee (Apis mellifera L.) colonies

Summary Honeybee nurses (8 days old) were injected with ¹⁴C-phenylalanine. These bees then dispensed the ¹⁴C-labelled protein-rich products of their hypopharyngeal glands to the queen and the brood, and also to young drones and workers of all age classes. In small colonies containing 400–800 bees, nearly one-quarter of the radioactivity which could not be recovered in the nurses was fed by them in a protein-bound form to other members of the worker caste. During one night, one nurse fed an average of 4–5 foragers with proteinaceous food. The role of nurses in the work allotment system of honeybee colonies therefore needs a new, extended definition. Nurses are largely responsible for preparing nutrients from pollen, which is difficult to digest. They then distribute the nutritionally valuable protein produced by their hypopharyngeal glands to practically all hive mates.Dedicated to Professor Dr. O. Kepka on the occasion of his 65th birthday     

Super and half-sister discrimination by honey bee workers (Apis mellifera L.) in a trophallactic bioassay

Summary Kin recognition and nepotism between honeybee workers (Apis mellifera L.) was analysed in a trophallactic bio-assay. Donor workers were fed dyed sugar syrup and introduced into a recipient group consisting of 12 to 15 workers of the same colony. After allowing for 1 hour of trophallaxis, the distribution of the dyed food was analysed with spectrophotometry. The subfamily composition in the recipient group was varied such that the donor bees had to discriminate between workers of 2 to 7 different patrilines. Donor bees preferentially fed super sisters if few patrilines were present in the recipient group. However, preferential feeding was not observed if the recipient group consisted of workers of more than three subfamilies. Since the natural degree of polyandry causes intracolonial genetic variance to exceed the genetic variability in the experiments, nepotistic behaviour among workers may not reveal intranidal subfamily recognition in honeybees.     

Octopamine receptors in the honeybee (Apis mellifera) brain and their disruption by RNA-mediated interference

Octopamine plays important neuromodulatory roles in the honeybee brain. Accordingly, mRNA from a recently identified honeybee octopamine receptor (AmOA1) is distributed throughout the brain. We have evaluated the occurrence of AmOA1 in the antennal lobe (AL) as well as rest of the brain (RB) by western blotting using an antiserum raised against a peptide selected from AmOA1 sequence. In addition to an expected band (78 kDa in the AL), one additional band (72 kDa) was identified from the AL and four bands (48, 60, 72 and 78 kDa) were observed in the RB. These bands were also recognized with antiserum against a different peptide segment from an octopamine receptor ortholog from the fruitfly (OAMB). Significant sequence identity with the peptide segment used to generate the antiserum was only found with OAMB and its splice variants in fruitfly; it was less conserved in other biogenic amine receptors from honeybee and other insects. Furthermore, western blot analysis performed on brains with dsRNA-treated antennal lobes showed a decrease in the intensity of all four bands. This suggests that AmOA1 antiserum specifically recognizes one or more types of AmOA1 receptors in the honeybee brain. We extend our earlier study of RNAi to quantify the rate of spread of dsRNA from a localized injection to other neuropils.     

Structural organization of ovarian follicle cells in the cotton bug (Dysdercus intermedius) and the honeybee (Apis mellifera)

Summary The somatic epithelia of Dysdercus and Apis follicles were analyzed by electron microscopy, and the patterns of F-actin and microtubules were studied by fluorescence microscopy. The epithelia in both species differ considerably in shape and in the organization of the cytoskeleton. During previtellogenic stages, the epithelium consists of columnar-shaped cells with small (Dysdercus) or no (Apis) lateral intercellular spaces. During vitellogenesis, the follicle cells round up; the intercellular spaces increase in size in Dysdercus follicles, whereas in Apis follicles they remain small. Along the basal surface of the follicle cells, there are conspicuous parallel bundles of microfilaments perpendicular to the anteroposterior axis of the follicles. In the honeybee, these microfilament bundles are present in long filopodia, most of which are embedded in thickenings of the basement membrane and extend over the surfaces of neighbouring cells. In the cotton bug, the basal surface of the follicle cells is thrown into parallel folds. The microfilament bundles are located just underneath the cell membrane where the folds contact the basement membrane. In the polar regions of the Dysdercus follicle, the epithelial cells become flat and adhere to each other without forming intercellular spaces. The basement membrane is particularly thick in the polar areas; this has also been observed in Apis follicles around the intercellular bridge connecting oocyte and nurse cells.     

Visual discrimination by the honeybee (Apis mellifera): the position of the common centre as the cue

Abstract. Bees can be trained to discriminate between a target with a 20° spot above a 10° spot of the same colour, and another target with the spots exchanged in position. Tests show that they do not remember the separate positions of spots of the same colour (including black) on the same target. The bees discriminate the difference in positions, in the vertical direction, of the common centres of the spots taken together, with or without green contrast.
Similar results are obtained in discriminations of a fixed T shape, each composed of two broad black bars subtending 8 by 24°, vs the same shape inverted. The trained bees fail to discriminate between the T shapes when the centroids are at the same level in the vertical direction. Moving the shapes in the horizontal direction in tests has less effect. Quite different results are obtained when the two bars of the T shape differ in colour. The bees discriminate the positions of the two colours separately, but they still fail to discriminate the shape of the T. The results can be explained by filters that detect the intensities within their fields, irrespective of shape, and weigh them according to their vertical angles from the horizontal midline. The normal function of these filters could be to detect the levels of objects relative to the horizon when the bee is in flight.     

The cys-loop ligand-gated ion channel superfamily of the honeybee, Apis mellifera

Members of the cys-loop ligand-gated ion channel (cys-loop LGIC) superfamily mediate neurotransmission in insects and are targets of successful insecticides. We have described the cys-loop LGIC superfamily of the honeybee, Apis mellifera, which is an important crop pollinator and a key model for social interaction. The honeybee superfamily consists of 21 genes, which is slightly smaller than that of Drosophila melanogaster comprising 23 genes. As with Drosophila, the honeybee possesses ion channels gated by acetylcholine, γ-amino butyric acid, glutamate and histamine as well as orthologs of the Drosophila pH-sensitive chloride channel (pHCl), CG8916, CG12344 and CG6927. Similar to Drosophila, honeybee cys-loop LGIC diversity is broadened by differential splicing which may also serve to generate species-specific receptor isoforms. These findings on Apis mellifera enhance our understanding of cys-loop LGIC functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. DQ667181–DQ667195.     

Selection on a haploid genotype for discrimination learning performance: Correlation between drone honey bees (Apis mellifera) and their worker progeny (Hymenoptera: Apidae)

Successful bidirectional selection for discriminative olfactory learning is reported for drone honey bees (Apis mellifera). Learning performance was evaluated using a discrimination conditioning procedure that required drones to discriminate between an appetitively reinforced odorant and one that was followed by punishment. Selective breeding produced high- and low-learning-performance lines of worker progeny that diverged from performance of workers whose fathers were selected at random. Furthermore, we show that levels of sucrose-induced sensitization are not correlated to learning performance. These results corroborate earlier findings and further demonstrate the power of selection on a haploid (drone) genotype. In addition, this study now shows that the demonstrated differences in learning performance cannot be completely accounted for by alteration of sucrose-induced sensitization. Thus, using this technique, it may be possible to select for associative conditioning without a pleiotropic increase in sensitization. The honey bee will be ideally suited to these types of correlation analyses in future studies.     

Patterns of chromatic information processing in the lobula of the honeybee, Apis mellifera L

The honeybee, Apis mellifera L., is one of the living creatures that has its colour vision proven through behavioural tests. Previous studies of honeybee colour vision has emphasized the relationship between the spectral sensitivities of photoreceptors and colour discrimination behaviour. The current understanding of the neural mechanisms of bee colour vision is, however, rather limited. The present study surveyed the patterns of chromatic information processing of visual neurons in the lobula of the honeybee, using intracellular recording stimulated by three light-emitting diodes, whose emission spectra approximately match the spectral sensitivity peaks of the honeybee. The recorded visual neurons can be divided into two groups: non-colour opponent cells and colour opponent cells. The non-colour opponent cells comprise six types of broad-band neurons and four response types of narrow-band neurons. The former might detect brightness of the environment or function as chromatic input channels, and the latter might supply specific chromatic input. Amongst the colour opponent cells, the principal neural mechanism of colour vision, eight response types were recorded. The receptive fields of these neurons were not centre surround as observed in primates. Some recorded neurons with tonic post-stimulus responses were observed, however, suggesting temporal defined spectral opponency may be part of the colour-coding mechanisms.     

Oogenesis in the honeybee Apis mellifera: cytological observations on the formation and differentiation of previtellogenic ovarian follicles

Summary Oogenesis is known to be important for embryonic pattern formation. For this reason we have studied the early differentiation of the honeybee ovariole histologically, ultrastructurally, and by staining F-actin with rhodaminyl-phalloidin. At the anterior tip of the ovariole, stem cells are lined up in a single file; they are organelle-poor but contain characteristic electrondense bodies with lysosomal properties. The presence of these bodies in cystocytes as well as prefollicle cells indicates that both cell types may be derived from the apical stem cells. During later stages of oogenesis, the follicle cells differentiate cytologically in different regions of the follicle. The organization of the intercellular bridges between cystocytes derived from a single cystoblast has been studied in detail. The polyfusomes in the intercellular bridges of cystocyte clusters stain with rhodaminyl-phalloidin and hence contain F-actin. Later, when the polyfusomes begin to desintegrate, F-actin rings form which line the rims of the intercellular bridges. Actin might be recruited from conspicuous F-actin stores which were detected in the germ-line cells. The F-actin rings are dissembled some time before the onset of vitellogenesis when the nurse chamber has grown to a length of about 200 m. At the basal side of the follicle cells (close to the basement membrane facing the haemocdele) parallel microfilament bundles encircle the ovariole. The microfilament bundles which are oriented mostly perpendicular to the long axis of the ovariole were first observed around the zone where the cystocyte divisions occur; after this phase the micro-filament bundles become organized differently in the follicle cells associated with the nurse cells and in the follicular epithelium of the oocyte. Correspondence to: H.O. Gutzeit     

Gene flow in admixed populations and implications for the conservation of the Western honeybee, Apis mellifera

Anthropogenic activity, especially modern apiculture, has considerable impact on the natural distribution of the Western honeybee, Apis mellifera, leading to the spread, replacement and fragmentation of many subspecies. This creates demand for the conservation of some subspecies, in particular, Apis mellifera mellifera, which once was widely distributed in Western Europe and nowadays is endangered through habitat loss and fragmentation. Moreover, A. m. mellifera may be further endangered by hybridisation in populations that now occur in artificial sympatry with other subspecies. Here, we quantify and compare individual hybridisation between sympatric and allopatric honeybee populations of A. m. mellifera and A. m. carnica using microsatellite markers and a Bayesian model-based approach. We had a special focus on pure breeding populations, which are a major tool in honeybee conservation. Our results demonstrate that subspecies are still highly differentiated, but gene flow is not prevented by the current management strategies, creating urgent demand for an improved conservation management of A. m. mellifera. However, the occurrence of a high number of pure individuals might suggest that some sort of hybrid barrier acts against the complete admixture of the two subspecies.     

Genetic pollution and number of matings in a black honey bee (Apis mellifera mellifera) population

Summary In the south-east of France, local honey bees possess only the B allele at the MDH locus, whereas the races which are usually imported into this area do not have this allele. The proportion of non-B genes in a sample of drones was used to measure the genetic pollution in the local population. Within the course of a breeding scheme of local bees, 99 queens, whose genotypes are BB, were naturally mated between April 25 and June 10, 1985 at la Tave (Gard, France). Twenty daughters-workers of each queen were analysed at the MDH locus. The frequency of the B allele in drones that mated with these queens is estimated by the proportion of workers with genotype BB and the genetic pollution by the cumulated frequency of the other alleles. The sampling variances of these frequencies involve a coefficient which is a function of the average number of drones mated with a queen. This latter parameter is estimated through the maximum likelihood method. In addition to the three well-known alleles, a rare allele (frequency=0.0055), possibly equivalent to the S1 allele described by Badino et al. (1983), has been found in three different colonies. Cumulating the frequencies of the non-B alleles results in an estimation of the genetic pollution equal to 0.0394 (±0.0071). This low value allows us to proceed to the next step of the selection project. The mean number of drones mated to a queen is 12.4 with a (10.4–19.3) confidence interval at the 90% level.     

Number of reproductive cycles of Varroa jacobsoni in honey-bee (Apis mellifera) colonies

Very little data exists concerning the number of reproductive cycles performed by individual Varroa mites. To understand the population dynamics of the Varroa mite it is necessary to know the number of fertile female offspring each Varroa female produces during her lifetime. The lifetime reproduction capacity of the mite consists of the mean number of fertile female offspring produced during each reproductive cycle multiplied by the mean number of cell passages. This paper describes an experimental design to estimate the number of reproductive cycles where mites are transferred to new mite-free colonies for reproduction in sealed brood cells. The data presented suggests that the mean number of reproductive cycles performed by the individual female mite is larger than previously accepted. Under optimal conditions, the mean number of reproductive cycles by Varroa females is probably greater than 1.5 but less than 2. Furthermore, the results show that the reproductive success of Varroa females going into cells to reproduce is not influenced by previous brood cycles.     

Ontogenesis of the mite Varroa jacobsoni Oud. in drone brood of the honeybee Apis mellifera L. under natural conditions

A study carried out during the summer of 1994, in southern England, investigated the developmental times and mortality ofVarroa jacobsoni inApis mellifera drone cells. The position and time of capping of 2671 naturally infested drone cells were recorded. Six hours after the cell was capped, 90% of the mites were free from the brood food to start feeding on the developing drone. The developmental time of the mite's first three female offspring (133±3 h) and the male offspring (150 h) and the intervals between egg laying (20–32 h) were similar to those found in worker cells. However, the mortality of the offspring was much lower in drone cells than worker cells. The mode numbers of eggs laid were six and five in drone and worker cells, respectively. All offspring had ample time to develop fully in drone cells with the sixth offspring reaching maturity approximately 1 day before the drone bee emerged. Normal mites (those which lay five or six viable eggs) produced on average four female adult offspring but since only around approximately 55% of the mite population produced viable offspring the mean number of viable adult female offspring per total number of mother mites was 2 to 2.2 in drone cells.     

Spatial memory, navigation and dance behaviour in Apis mellifera

Navigation and dance communication in Apis mellifera have been extensively studied on the level of sensory processing, but the structure and content of the spatial memory underlying such phenomena have yet to be addressed. Here we survey new findings indicating that the memory used by bees to navigate within the range of their orientation flights is much more complex than hitherto thought. It appears to allow them to decide between at least two goals in the field, and to steer towards them over considerable distances. Two models concerning the structure of bees’ spatial memory are developed from new empirical evidence. The first one relies on the integration of at least two flight vectors, while the second assumes the existence of a ‘functional’ map based on the information available on-site. These findings also raise questions about the process of encoding and decoding information in the context of the waggle dance. We review published data and recent evidence indicating that memories of topographical features might also be involved in dance communication, and point out what needs to be addressed to elucidate the corresponding memory demands. The flight paths of recruited bees can now be traced by means of radar techniques, and thus tools are available to tackle these questions.     

Distribution of Nosema ceranae in the European honeybee, Apis mellifera in Japan

The microsporidian species, Nosema apis and Nosema ceranae are both known to infect the European honeybee, Apis mellifera. Nosema disease has a global distribution and is responsible for considerable economic losses among apiculturists. In this study, 336 honeybee samples from 18 different prefectures in Japan were examined for the presence of N. apis and N. ceranae using a PCR technique. Although N. ceranae was not detected in most of the apiaries surveyed, the parasite was detected at three of the sites examined. Further, N. ceranae appears to be patchily distributed across Japan and no apparent geographic difference was observed among the areas surveyed. In addition, the apparent absence of N. apis suggests that N. ceranae may be displacing N. apis in A. mellifera in Japan. Partial SSU rRNA gene sequence analysis revealed the possible existence of two N. ceranae groups from different geographic regions in Japan. It seems likely that these microsporidian parasites were introduced into Japan through the importation of either contaminated honeybee-related products or infected queens. This study confirmed that PCR detection is effective for indicating the presence of this pathogen in seemingly healthy colonies. It is therefore hoped that the results presented here will improve our understanding of the epidemiology of Nosema disease so that effective controls can be implemented.