Recognition of a familiar place by the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Recent work shows that at any one place bees detect a limited variety of simple cues in parallel. At each choice point, they recognize a few cues in the range of positions where the cues occurred during the learning process. There is no need to postulate that they re-assemble the surrounding panorama in memory; only that they retain memories of the coincidences of cues in the expected retinotopic directions. The cues could be stimuli that excite groups of peripheral visual neurons. All the experimentally known cues are described, including modulation of the receptors, the locations of areas of black or colour, the nearness, size, averaged edge orientation, and radial and tangential edges. Cues of each type are separately summed within large fields, the size of which varies with the cue. Local orientation cues from edges at right angles cancel each other within each field, which also suggests that the discrimination of shape and texture is limited. Resolution depends on lateral interactions and the number of ommatidia required for each cue. To identify a new place, a few sparse cues, together with their directions, are learned in orientation flights. When the bee returns, the cues in the panorama are progressively matched as they coincide with the cues in memory. The limited number of cues, though economical for memory, may restrict the foraging behaviour and lead to flower constancy. This kind of a visual system is a candidate model for other animals or machines with economical processing systems.     

Expression of <Emphasis Type="Italic">Pax group III</Emphasis> genes in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Pax group III genes are involved in a number of processes during insect segmentation. In Drosophila melanogaster, three genes, paired, gooseberry and gooseberry-neuro, regulate segmental patterning of the epidermis and nervous system. Paired acts as a pair-rule gene and gooseberry as a segment polarity gene. Studies of Pax group III genes in other insects have indicated that their expression is a good marker for understanding the underlying molecular mechanisms of segmentation. We have cloned three Pax group III genes from the honeybee (Apis mellifera) and examined their relationships to other insect Pax group III genes and their expression patterns during honeybee segmentation. The expression pattern of the honeybee homologue of paired is similar to that of paired in Drosophila, but its expression is modulated by anterior–posterior temporal patterning similar to the expression of Pax group III proteins in Tribolium. The expression of the other two Pax group III genes in the honeybee indicates that they also act in segmentation and nervous system development, as do these genes in other insects.     

Individual recruitment in honeybees, <Emphasis Type="Italic">Apis mellifera</Emphasis> L.

The recruitment of honeybee foragers individually exploiting a low-flow rate-feeder that presented different temporal reward programs was experimentally analyzed. By capturing hive bees that landed at the feeder in a 2-h period, the arrival rate of incoming bees could be obtained. With this procedure we quantitatively analyzed the maximum number of hive bees that can be brought to the feeding station by single foragers. Test bees collected sucrose solution during 12 visits to a rate-feeder located 160 m from the hive. The constant programs offered 0.6, 1.2, or 2.4 M sugar for all 12 visits, while the variable programs delivered either 0.6, 1.2, or 0.6 M or 0.6, 2.4, or 0.6 M, with four visits for each molarity. Results showed that the sucrose concentration exploited by single foragers increased the arrival rate. Moreover, there was a linear relationship within this range of sucrose concentrations that presented a slope of 1.58. Since the sugar solutions were provided at the same flow rate (5 μl/min) in all the programs, the arrival rate expressed in terms of sucrose flow rate (milligrams of sucrose/minute) shows that one additional incoming bee per hour arrived when the single forager assessed an increase in the sucrose flow rate of 0.75 mg sucrose/min at the rate-feeder. The absence of differences in the frequency of visits of the single foragers during the constant programs suggests that the differences observed in the arrival rate can mainly be explained by a more intensive display of the recruitment mechanisms performed per foraging trip instead of by their iterativeness throughout different foraging cycles. Variable reward programs showed that arrival rate is rapidly adjusted according to the reward change and is independent of its magnitude. Received in revised form: 17 August 2001 Electronic Publication     

The effect of complexity on the discrimination of oriented bars by the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Visual discrimination of black bars by honeybees was studied in a Y-choice apparatus with fixed vertical patterns at constant range. The problem is to discover how bees remember different degrees of complexity of the orientation cue. Previous conclusions with parallel gratings and single bars disagree. With broad bars versus orthogonal bars, the bees learn the orientation cue if the bars are centred at the same place, but they learn the position cue in the vertical direction when the bars are at different places on the two targets. With several bars on each target, the bees learn their orientation and positions. As fixed patterns increase in complexity, the bees follow a simple rule, to look only at the range of places where the cues were displayed. The frame of reference is disrupted when a black spot is added to the training pattern. There is abundant evidence that the bees do not re-assemble the pattern or learn shapes. The filters that detect the position and orientation cues are coarsely tuned, so that they respond in a graded way, but the memory of the range of directions of the cue, as seen from the point of choice, is more exact.     

Pupal developmental temperature and behavioral specialization of honeybee workers (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.)

Honeybees (Apis mellifera) are able to regulate the brood nest temperatures within a narrow range between 32 and 36°C. Yet this small variation in brood temperature is sufficient to cause significant differences in the behavior of adult bees. To study the consequences of variation in pupal developmental temperature we raised honeybee brood under controlled temperature conditions (32, 34.5, 36°C) and individually marked more than 4,400 bees, after emergence. We analyzed dancing, undertaking behavior, the age of first foraging flight, and forager task specialization of these workers. Animals raised under higher temperatures showed an increased probability to dance, foraged earlier in life, and were more often engaged in undertaking. Since the temperature profile in the brood nest may be an emergent property of the whole colony, we discuss how pupal developmental temperature can affect the overall organization of division of labor among the individuals in a self-organized process.     

New approach to the mitotype classification in black honeybee <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> and Iberian honeybee <Emphasis Type="Italic">Apis mellifera iberiensis</Emphasis>

The black honeybee Apis mellifera mellifera L. is today the only subspecies of honeybee which is suitable for commercial breeding in the climatic conditions of Northern Europe with long cold winters. The main problem of the black honeybee in Russia and European countries is the preservation of the indigenous gene pool purity, which is lost as a result of hybridization with subspecies, A. m. caucasica, A. m. carnica, A. m. carpatica, and A. m. armeniaca, introduced from southern regions. Genetic identification of the subspecies will reduce the extent of hybridization and provide the gene pool conservation of the black honeybee. Modern classification of the honeybee mitotypes is mainly based on the combined use of the DraI restriction endonuclease recognition site polymorphism and sequence polymorphism of the mtDNA COI–COII region. We performed a comparative analysis of the mtDNA COI–COII region sequence polymorphism in the honeybees of the evolutionary lineage M from Ural and West European populations of black honeybee A. m. mellifera and Spanish bee A. m. iberiensis. A new approach to the classification of the honeybee M mitotypes was suggested. Using this approach and on the basis of the seven most informative SNPs of the mtDNA COI–COII region, eight honeybee mitotype groups were identified. In addition, it is suggested that this approach will simplify the previously proposed complicated mitotype classification and will make it possible to assess the level of the mitotype diversity and to identify the mitotypes that are the most valuable for the honeybee breeding and rearing.     

The functions of antennal mechanoreceptors and antennal joints in tactile discrimination of the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.)

Honeybees learn and discriminate excellently between different surface structures and different forms of objects, which they scan with their antennae. The sensory plate on the antennal tip plays a key role in the perception of mechanosensory and gustatory information. It is densely covered with small tactile hairs and carries a few large taste hairs. Both types of sensilla contain a mechanoreceptor, which is involved in the antennal scanning of an object. Our experiments test the roles of the mechanoreceptors on the antennal tip in tactile antennal learning and discrimination. Joints between head capsule and scapus and between scapus and pedicellus enable the bee to perform three-dimensional movements when they scan an object. The role of these joints in tactile antennal learning and discrimination is studied in separate experiments. The mechanoreceptors on the antennal tip were decisive for surface discrimination, but not for tactile acquisition or discrimination of shapes. When the scapus–pedicellus joint or the headcapsule–scapus joint was fixed on both antennae, tactile learning was still apparent but surface discrimination was abolished. Fixing both scapi to the head capsule reduced tactile acquisition.     

The pheromones of laying workers in two honeybee sister species: <Emphasis Type="Italic">Apis cerana</Emphasis> and <Emphasis Type="Italic">Apis mellifera</Emphasis>

When a honeybee colony loses its queen, workers activate their ovaries and begin to lay eggs. This is accompanied by a shift in their pheromonal bouquet, which becomes more queen like. Workers of the Asian hive bee Apis cerana show unusually high levels of ovary activation and this can be interpreted as evidence for a recent evolutionary arms race between queens and workers over worker reproduction in this species. To further explore this, we compared the rate of pheromonal bouquet change between two honeybee sister species of Apis cerana and Apis mellifera under queenright and queenless conditions. We show that in both species, the pheromonal components HOB, 9-ODA, HVA, 9-HDA, 10-HDAA and 10-HDA have significantly higher amounts in laying workers than in non-laying workers. In the queenright colonies of A. mellifera and A. cerana, the ratios (9-ODA)/(9-ODA + 9-HDA + 10-HDAA + 10-HDA) are not significantly different between the two species, but in queenless A. cerana colonies the ratio is significant higher than in A. mellifera, suggesting that in A. cerana, the workers’ pheromonal bouquet is dominated by the queen compound, 9-ODA. The amount of 9-ODA in laying A. cerana workers increased by over 585% compared with the non-laying workers, that is 6.75 times higher than in A. mellifera where laying workers only had 86% more 9-ODA compared with non-laying workers.     

L-Glutamate in formation of long-term memory in the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Participation of metabotropic glutamate receptors (mGluR) in associative learning of the honeybee Apis mellifera was studied in behavioral and electrophysiological experiments with use of pharmacological analysis. By the method of conditioned reflexes, effects of systemic injections of mGluR agonists and antagonists were studied on retention of elaborated conditioned reflexes in short-term and long-term memory. Injection of aminocyclopentandicarbonic acid (ACPD), ibotenate, and phosphoserine stimulated the memory in 3 h after the single learning procedure. The long- term memory was inhibited by mGluR antagonists — methylcarboxyphenylglycine and amino- phosphonopropionate as well as by non-competitive antagonists of mGluR₁(CPCCOEt) and of mGluR₅ (SIB 1757). Electrophysiological experiments demonstrated changes of characteristics of the action potential recorded from mushroom bodxies in response to stimulation of antennal lobes and during injections of mGluR III phosphoserine and the mGluR₁ antagonist CPCCOEt. The obtained data allow suggesting participation of different groups of metabotropic glutamate receptors in the process of the honeybee associative learning.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 40, No. 6, 2004, pp. 539–545.     

Vitellogenin transcytosis in follicular cells of the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis> and the wasp <Emphasis Type="Italic">Polistes simillimus</Emphasis>

Vitellogenin receptor (VgR) is a low-density lipoprotein receptor responsible for the mediated endocytosis of vitellogenin (Vg) during egg formation in insects. The maturing oocyte is enveloped by a follicular epithelium, which has large intercellular spaces during Vg accumulation (patency). However, Vg has been reported in the cytoplasm of follicular cells, indicating that there may be a transcellular route for its transport. This study verified the presence of VgR in the follicular cells of the ovaries of the honeybee Apis mellifera and the wasp Polistes simillimus in order to evaluate if Vg is transported via transcytosis in these insects. Antibodies specific for vitellogenin receptor (anti-VgR), vitellogenin (anti-Vg), and clathrin (anti-Clt) were used for immunolocalization. The results showed the presence of VgR on the apical and basal plasma membranes of follicular cells of the vitellogenic follicles in both species, indicating that VgR may have been transported from the basal to the apical cell domain, followed by its release into the perivitelline space, evidenced by the presence of apical plasma membrane projections containing VgR. Co-localization proved that Vg bind to VgR and that the transport of this protein is mediated by clathrin. These data suggest that, in these social insects, Vg is transported via clathrin-mediated VgR transcytosis in follicular cells.     

Influences of octopamine and juvenile hormone on locomotor behavior and <Emphasis Type="Italic">period</Emphasis> gene expression in the honeybee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

Octopamine (OA) and juvenile hormone (JH) are implicated in the regulation of age-based division of labor in the honeybee, Apis mellifera. We tested the hypothesis that these two neuroendocrine signals influence task-associated plasticity in circadian and diurnal rhythms, and in brain expression of the clock gene period (per). Treatment with OA, OA antagonist (epinastine), or both, did not affect the age at onset of circadian rhythmicity or the free running period in constant darkness (DD). Young bees orally treated with OA in light–dark (LD) illumination regime for 6 days followed by DD showed reduced alpha (the period between the daily onset and offset of activity) during the first 4 days in LD and the first 4 days in DD. Oral treatment with OA, epinastine, or both, but not manipulations of JH levels, caused increased average daily levels and aberrant patterns of brain per mRNA oscillation in young bees. These results suggest that OA and JH do not influence the development or function of the central pacemaker but rather that OA influences the brain expression of a clock gene and characteristics of locomotor behavior that are not thought to be under direct control of the circadian pacemaker. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Molecular determinants of caste differentiation in the highly eusocial honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Background  

In honeybees, differential feeding of female larvae promotes the occurrence of two different phenotypes, a queen and a worker, from identical genotypes, through incremental alterations, which affect general growth, and character state alterations that result in the presence or absence of specific structures. Although previous studies revealed a link between incremental alterations and differential expression of physiometabolic genes, the molecular changes accompanying character state alterations remain unknown.     

Prepro-tachykinin gene expression in the brain of the honeybee<Emphasis Type="Italic"> Apis mellifera</Emphasis>

We have recently identified a tachykinin-related peptide (AmTRP) from the mushroom bodies (MBs) of the brain of the honeybee Apis mellifera L. by using direct matrix-assisted laser desorption/ionization with time-of-flight mass spectometry and have isolated its cDNA. Here, we have examined prepro-AmTRP gene expression in the honeybee brain by using in situ hybridization. The prepro-AmTRP gene is expressed predominantly in the MBs and in some neurons located in the optic and antennal lobes. cDNA microarray studies have revealed that AmTRP expression is enriched in the MBs compared with other brain regions. There is no difference in AmTRP-expressing cells among worker, queen, and drone brains, suggesting that the cell types that express the prepro-AmTRP gene do not change according to division of labor, sex, or caste. The unique expression pattern of the prepro-AmTRP gene suggests that AmTRPs function as neuromodulators in the MBs of the honeybee brain.This work was supported by a Grant-in-Aid from the Bio-oriented Technology Research Advancement Institution (BRAIN)     

Analysis of RNA Silencing in Agroinfiltrated Leaves of <Emphasis Type="Italic">Nicotiana Benthamiana</Emphasis> and <Emphasis Type="Italic">Nicotiana Tabacum</Emphasis>

In this study we analyse several aspects of cytoplasmic RNA silencing by agroinfiltration of DNA constructs encoding single- and double-stranded RNAs derived from a GFP transgene and from the endogenous Virp1 gene. Both types of inductors resulted after 2–4 days in much higher concentration of siRNAs in the agroinfiltrated zone than normally seen during systemic silencing. More specifically, infiltration of two transgene hairpin constructs resulted in elevated levels of siRNAs. However, differences between the two constructs were observed: the antisense–sense arrangement was more effective than the sense–antisense order. For both double-stranded forms, we observed a relative increase of the 24-mer size class of siRNAs. When a comparable hairpin construct of the endogenous Virp1 gene was assayed, the portion of the 24-mer siRNA class remained low as observed for all kinds of single-stranded inducers. The lack of increase of Virp1-derived 24-mers was independent of the expression level, as demonstrated by agroinfiltration into a transgenic plant that overexpressed Virp1 and showed the same pattern. Using transducer constructs, we could detect within a week transitive silencing from GFP to GUS sequences in the infiltrated zone and in either direction 5′–3′ and 3′–5′. Conversely, for the endogenous Virp1 gene neither transitive silencing nor the induction of systemic silencing could be observed. These results are discussed in view of the current models of RNA silencing.     

Local honeybee (<Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> L.) populations in the Urals

The COI-COII intergenic region of mitochondrial DNA (mtDNA) was studied in local honeybee (Apis mellifera mellifera) L. populations from the Middle and Southern Urals. Analysis of bee colonies in these regions revealed apiaries enriched in families descending from A. m. mellifera in the maternal lineage. These results confirm the suggestion of preservation of A. m. mellifera refuges in the Urals and provide grounds for work on the preservation of the gene pool of this bee variety, valuable for all Russia.     

Polymorphism analysis of <Emphasis Type="Italic">csd</Emphasis> gene in six <Emphasis Type="Italic">Apis mellifera</Emphasis> subspecies

The complementary sex determination (csd) gene is the primary gene determining the gender of honey bees (Apis spp). In this study we analyzed the polymorphism of csd gene in six Apis mellifera subspecies. The genomic region 3 of csd gene in these six A. mellifera was cloned, and identified. A total of 79 haplotypes were obtained from these six subspecies. Analysis showed that region 3 of csd gene has a high level of polymorphism in all the six A. mellifera subspecies. The A. m. anatolica subspecies has a slightly higher nucleotide diversity (π) than other subspecies, while the π values showed no significant difference among the other five subspecies. The phylogenetic tree showed that all the csd haplotypes from different A. mellifera subspecies are scattered throughout the tree, without forming six different clades. Population differentiation analysis showed that there are significant genetic differentiations among some of the subspecies. The NJ phylogenetic tree showed that the A. m. caucasica and A. m. carnica have the closest relationship, followed by A. m. ssp, A. m. ligustica, A. m. carpatica and A. m. anatolica that were gathered in the tree in turn.     

Seven genes of mitochondrial genome enabling differentiation of honeybee subspecies <Emphasis Type="Italic">Apis mellifera</Emphasis>

On the basis of comparative sequence analysis of 12 honeybee mitochondrial genes, seven genes enabling us to differentiate honeybees subspecies of the A, M, C, O evolutionary lineages were found. Applying comparative sequence analysis of ND2 gene mtDNA as an example on a statistically valid sample size, we showed a high level of differentiating ability of this gene and assumed that each of these seven genes probably can be used for differentiation of the subspecies within four evolutionary lineages.