Worker honey bee ovary development: seasonal variation and the influence of larval and adult nutrition

We examined the effect of larval and adult nutrition on worker honey bee (Apis mellifera L.) ovary development. Workers were fed high or low-pollen diets as larvae, and high or low-protein diets as adults. Workers fed low-protein diets at both life stages had the lowest levels of ovary development, followed by those fed high-protein diets as larvae and low- quality diets as adults, and then those fed diets poor in protein as larvae but high as adults. Workers fed high-protein diets at both life stages had the highest levels of ovary development. The increases in ovary development due to improved dietary protein in the larval and adult life stages were additive. Adult diet also had an effect on body mass. The results demonstrate that both carry-over of larval reserves and nutrients acquired in the adult life stage are important to ovary development in worker honey bees. Carry-over from larval development, however, appears to be less important to adult fecundity than is adult nutrition. Seasonal trends in worker ovary development and mass were examined throughout the brood rearing season. Worker ovary development was lowest in spring, highest in mid-summer, and intermediate in fall.     

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies.     

Worker-worker inhibition of honey bee behavioural development independent of queen and brood

Summary. Foragers inhibit the behavioural development of young adult worker honey bees, delaying the age at onset of foraging. But the similar effect caused by pheromones produced by both the queen and brood raised the possibility that some of the previously attributed forager effects might be due to queen, brood, or both. Here we studied whether physical contacts between young bees and old foragers can inhibit behavioural development while controlling for queen and brood effects. Results demonstrated that foragers inhibit the behavioural development of young adult worker bees independent of the queen and brood, via a mechanism that requires physical contact.Received 24 November 2003; revised 27 March 2004; accepted 21 April 2004.     

Sex-specific developmental profiles of juvenile hormone synthesis in honey bee larvae

Summary Juvenile hormone synthesis in drone larvae of the honey bee was measured by an in vitro radiochemical assay. The developmental profile of corpora allata activity in male larvae showed considerable differences from queen larvae, the presumptive reproductive females, and was comparable to workers, the sterile female morph. Drone and worker larvae, however, differed drastically in the regulation of juvenile hormone biosynthesis, as revealed by the addition of farnesoic acid to the culture medium. This precursor stimulated juvenile hormone synthesis of drone glands nearly eightfold, whereas in worker larvae it is known to lead to an accumulation of methyl farnesoate. The sex-specific differences in endocrine activity indicate a role for juvenile hormone in the expression of genetically determined sexually dimorphic characters during metamorphosis, a role not currently accounted for in models describing endocrine regulation of insect development. Correspondence to: K. Hartfelder     

Germ cell cluster formation and cell death are alternatives in caste-specific differentiation of the larval honey bee ovary

Summary

Caste-specific differentiation of the female honey bee gonad takes place in the fifth larval instar. In queen larvae most ovarioles exhibit almost simultaneous formation of numerous germ cell clusters within the first 20 h after the last larval molt. Ultrastructurally distinctive fusomal cytoplasm connects these cystocytes. Germ cell differentiation is accompanied by morphological changes in somatic components of the ovarioles, the follicle and the terminal filament cells. Subsequently, queen ovarioles elongate and differentiate basal stalks that coalesce in a basal calyx. A second round of mitotic activity was found to occur in the late prepupal and early pupal queen ovary. This round may elevate germ cell numbers composing each cluster to levels observed in follicles of adult honey bee queens. In contrast, germ cell cluster formation does not occur in most of the 120–160 ovarioles of the larval worker ovary, but instead many cells in such ovarioles show signs of impending degeneration, such as large autophagic bodies. DNA extracted from worker ovaries did not reveal nucleosomal laddering, and ultrastructurally, chromatin in germ cell nuclei appeared intact. In the 4–7 surviving ovarioles of the small worker ovary, germ cell clusters were found with ultrastructural characteristics identical to those in queen ovarioles. The temporal window during which divergence in developmental pathways of the larval ovaries initiates shortly after the last larval molt coincides with caste-specific differences in juvenile hormone titer which have long been considered critical to caste-specific morphogenesis.     

Factors influencing host choice of the honey bee parasite Varroa jacobsoni Oud

Reproducing Varroa jacobsoni obtained from brood cells of Apis mellifera L. with 13–16 day old bees (pupae) and Varroa mites kept on adult bees for at least 8 days were simultaneously tested for their choice in three host types. Comparisons were made of attractiveness of Varroa jacobsoni to nurse bees, pollen foragers as to larvae from nearly capped brood cells. Host choices were observed in Petri dishes and in an Y-shaped olfactometer. Varroa jacobsoni obtained from capped brood cells showed a stronger preference for nurse bees in Petri dish simultaneous choice tests with pollen foragers or larvae than did mites which were previously kept on adult bees. In olfactometer simultaneous choice tests, the two mite test groups showed no clear difference in preferences for bees of different ages. The preference of Varroa jacobsoni for bees of different ages is therefore not only influenced by host factors but also by intrinsic factors in female mites that depend on the mite's reproductive stage.     

Behaviour of Varroa mites invading honey bee brood cells

Invasion behaviour of Varroa jacobsoni into honey bee brood cells was studied using an observation hive. The mites were carried close to a suitable brood cell by the bees. Subsequently, the mites moved from the bees to the rim of the cell, walked quickly inside, crawled between the larva and the cell wall, and moved onto the bottom of the cell. Varroa mites were never seen walking across the comb, and entering and leaving brood cells as has been described for Tropilaelaps clareae. Differences in invasion strategies between V. jacobsoni and T. clareae are discussed.     

Self-assemblage formation in a social insect: the protective curtain of a honey bee swarm

Summary. This paper considers a little-studied topic in the biology of social insects: the formation of self-assemblages. It focuses on the mechanisms whereby the outermost workers in a bivouacked swarm of honey bees, when rained upon, form a water repellent curtain of bees over the swarm cluster. Specifically, we analyzed how the worker bees in the mantle of a swarm cluster adjust their body orientation, wing spread, and inter-individual spacing to form a protective curtain when wetted. When warm and dry, the mantle bees orient their bodies weakly with respect to gravity, do not tuck their heads under adjacent bees, have high variability in wing spread, and space themselves widely. In contrast, when warm and wet, the mantle bees orient uniformly with head upward, tuck their heads beneath the abdomens of bees above, hold their wings together, and press tightly together. This produces a surface that closely resembles a tiled roof. When cool and dry, the mantle bees generally orient their bodies with head upward, press their heads into the interior of the cluster, hold their wings wide apart, and draw close together. We also examined the age distribution of the mantle bees. Older bees are more likely than younger bees to be found in the mantle of a swarm, perhaps because younger bees are more important than older bees to colony survival after swarming and so occupy a more sheltered position in a swarm. Finally, we tested whether swarm clusters that have formed a protective curtain shed water more effectively than ones that have not formed a curtain. We found that this is the case.Received 28 November 2003; revised 29 February 2004; accepted 11 April 2004.     

Effect of age,behaviour and social environment on honey bee brain plasticity

We examined the effects of behaviour, age and social environment on mushroom body volume in adult bees. The mushroom bodies are regions of the central brain important for sensory integration and learning. Their volume was influenced by behaviour throughout life: always larger in forager bees than age-matched nurse bees, even in old bees up to 93 days of age as adults. Mushroom body development was influenced by the social environment in the first 8 days of adult life, with different environments having markedly different effects on mushroom body size. Compared to hive-reared bees, isolation slowed mushroom body growth, but bees reared in isolation confined with a single dead bee showed a dramatic increase in mushroom body volume comparable to that seen in active foragers. Despite their precocious mushroom body development, these bees did not show improved performance in an olfactory learning test. Since simple environmental manipulations can both accelerate and delay mushroom body growth in young bees, and since mushroom body volume is sensitive to behaviour throughout life, the honey bee has great potential as a model for exploring the interactions between environment, behaviour and brain structure.     

Endocrine modulation of a pheromone-responsive gene in the honey bee brain

Pheromones cause dramatic changes in behavior and physiology, and are critical for honey bee colony organization. Queen mandibular pheromone (QMP) regulates multiple behaviors in worker bees (Slessor et al. in J Chem Ecol 31(11):2731–2745, 2005). We also identified genes whose brain expression levels were altered by exposure to QMP (Grozinger et al. in Proc Natl Acad Sci USA 100(Suppl 2):14519–14525, 2003). Krüppel-homolog 1 (Kr-h1) RNA levels were significantly downregulated by QMP, and were higher in foragers than in nurses (Whitfield et al. in Science 302(5643):296–299, 2003). Here we report on results of behavioral and pharmacological experiments that characterize factors regulating expression of Kr-h1. Foragers have higher brain levels of Kr-h1 than in-hive bees, regardless of age and pheromone exposure. Furthermore, forager Kr-h1 levels were not affected by QMP. Since the onset of foraging is caused, in part, by increasing juvenile hormone blood titers and brain octopamine levels, we investigated the effects of octopamine and methoprene (a juvenile hormone analog) on Kr-h1 expression. Methoprene produced a marginal (not significant) increase in Kr-h1 expression, but Kr-h1 brain levels in methoprene-treated bees were no longer downregulated by QMP. Octopamine did not modulate Kr-h1 expression. Our results demonstrate that the gene expression response to QMP is not hard-wired in the brain but is instead dependent on worker behavioral state.     

Colour association influences honey bee choice between sucrose concentrations

Certain colours associated with floral food resources are more quickly learned by honey bees (Apis mellifera) than are other colours. But the impact of colour, and other floral cues, on bee choice behaviour has not yet been determined. In these experiments, colour association and sugar concentration of reward were varied to assess how they interact to affect bee choice behaviour. Thirty-five bees were individually given binary choices between blue and yellow artificial flowers that contained either the same rewards or rewards of different sucrose concentrations. Honey bee choice between sucrose concentrations was affected by colour association and this effect was greatest when absolute difference between rewards was the lowest. The honey bee's ability to maximize energetic profitability during foraging is constrained by floral cue effectiveness.     

Patterns of inheritance with RAPD molecular markers reveal novel types of polymorphism in the honey bee

Summary The polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 11 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.     

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 influences honey bee foraging preference

Colony condition and differences in individual preferences influence forage type collected by bees. Physiological bases for the changing preferences of individual foragers are just beginning to be examined. Recently, for honey bees octopamine is shown to influence age at onset of foraging and probability of dance for rewards. However, octopamine has not been causally linked with foraging preference in the field. We tested the hypothesis that changes in octopamine may alter forage type (preference hypothesis). We treated identified foragers orally with octopamine or its immediate precursor, tyramine, or sucrose syrup (control). Octopamine-treated foragers switched type of material collected; control bees did not. Tyramine group results were not different from the control group. In addition, sugar concentrations of nectar collected by foragers after octopamine treatment were lower than before treatment, indicating change in preference. In contrast, before and after nectar concentrations for bees in the control group were similar. These results, taken together, support the preference hypothesis.     

Effect of acaricide resistance on reproductive ability of the honey bee mite Varroa destructor

The reproduction of pyrethroid-resistant Varroa destructor mite, a brood parasite of honey bees, was observed in Weslaco, Texas, and the results compared with known susceptible mite populations from other studies. Seven Apis mellifera colonies that had mite populations resistant to the acaricide Apistan^™ were used. Pyrethroid-resistance was confirmed when only 17% rather than 90% of mites confined in dishes containing Apistan^™ died after 12 h of exposure. The average number of eggs laid by resistant mites invading worker and drone cells was 4.4 and 5.4 respectively. This is similar to the number of eggs laid by susceptible mites in worker (4.4–4.8) or drone (4.7–5.5) cells. Also the average number of fertilised V. destructor female mites produced by resistant mites in worker (1.0) and drone (2.1) cells were similar to the number produced by susceptible mites in worker (0.9) and drone (1.9–2.2) cells. In addition, no major differences between the resistant and susceptible mite populations were observed in either worker or drone cells when six different reproductive categories and offspring mortality rates were compared. Therefore, it appears that there is little or no reproductive fitness cost associated with pyrethroid resistance in V. destructor in Texas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Flight of the honey bee VII: metabolic power versus flight speed relation

The existing experimental data on metabolic power P _m of honey bees are critically discussed, partly corrected for real flight conditions and plotted as a function of flight speed v. New wind tunnel measurements of tethered flight under near-natural conditions are added in the range 3.3<v<5.1 m·s^-1, derived from exhaustion flight measurements. Within this small sector the latter measurements can be characterised by a linear correlation: P _m(mW)=6.72v (m·s^-1)+13.83, the slope of which is significantly different from zero. The over-all P _m(v) curve is significantly not a straight line of zero slope but a U-shaped minimum curve and may be approximated by a second-order polynom: P _m=49.2-8.9v+1.5v ². The same is true for relative metabolic power, P _{m rel (e)} related to empty body mass of 76.5 mg: P _{m rel(e)}=630.0-114.0v+19.2v ² (P _m in mW: P _{m rel} in mW·g^-1; v in m·s^-1). The data support the existence of a U-shaped power-versus-speed curve in bees.Abbreviations bm body mass (mg) - f full - e empty - mu muscles - P _m (mJ·s^-1=mW) metabolic power (input) - P _{m rel} (mW·g^-1) relative metabolic power - P _mec (mW) mechanical power (output) - efficiency (of the flight musculature) - t(s) flight time - v (m·s^-1) relative speed between bee and air     

Polar tube protein gene diversity among Nosema ceranae strains derived from a Greek honey bee health study

Honey bee samples from 54 apiaries originating from 37 geographic locations of Greece were screened for Nosema apis and Nosema ceranae. Furthermore 15 samples coming from 12 geographic locations were screened also for Paenibacilluslarvae and Melissococcus plutonius and seven honey bee virus species, for the first time on a nation-wide level. There was a tendency in finding proportionally higher spore counts in samples from apiaries that suffered important colony losses. P. larvae bacteria were identified in two samples and each of the tested bee viruses could be detected in at least one of the examined samples, with IAPV, CBPV and SBV being the least abundant and BQCV and DWV being the most abundant. In the study we focused on polymorphism of a N. ceranae gene encoding a polar tube protein (PTP) as similar genes were proven to be highly polymorphic in the microsporidian parasites Encephalitozoon cuniculi and Encephalitozoon hellem. The polymorphism observed in the PTP gene sequences from a single sample (bee hive) was unexpected and can thus be considered to be a major obstacle for genotyping.     

Negative correlation between Nosema ceranae spore loads and deformed wing virus infection levels in adult honey bee workers

Interactions between pathogens might contribute to honey bee colony losses. Here we investigated if there is an association between the microsporidian Nosema ceranae and the deformed wing virus (DWV) in different body sections of individual honey bee workers (Apis mellifera ligustica) under exclusion of the vector Varroa destructor. Our data provide correlational evidence for antagonistic interactions between the two pathogens in the midgut of the bees.     

Testing the blank slate hypothesis: why honey bee colonies accept young bees

Summary Special features facilitate the admission of new members, such as neonates, to otherwise closed animal societies. In eusocial insects, such as honeybees and paper wasps, young adults acquire a colony recognition phenotype from other colony members or nesting materials. Older adults must exempt them from expulsion during the acquisition period. Newly emerged adult honeybees gain tolerance in their colony before their acquisition of the colony recognition phenotype by presenting a blank slate, absent recognition cues. This makes them generically acceptable in honey bee colonies. This strategy is analogous to the easily recognizable phenotypes associated with juvenility in birds and mammals.Received 25 September 2002; revised 20 June 2003; accepted 2 July 2003.