Ecdysteroid titer and reproduction in queens and workers of the honey bee and of a stingless bee: loss of ecdysteroid function at increasing levels of sociality?

Evidence from field wasps and bumblebees appoints the endocrine system as a mediator between dominance status and ovarian activity in primitively social Hymenoptera. In this comparative study on ecdysteroid titers in the highly social honey bee, Apis mellifera, and a stingless bee, Melipona quadrifasciata, we focussed on the relationship between the ecdysteroid titer, social conditions (presence or absence of the queen), and ovary activity. In contrast to bumblebees, ecdysteroid titers in honey bee and stingless bee workers were either not altered, or dropped to even lower levels after the queen was removed. We also did not detect differences between virgin queens and mated, egg laying queens. These results suggest that ecdysteroids may have lost most of their reproductive functions - yet gained functions in larval caste differentiation - as higher levels of social organization were attained in the evolution of social insects. The observation that ecdysteroid titers are transiently elevated in young workers adds a new, yet functionally still speculative facet to hormonal regulation in insect societies.     

Pathological effects of the microsporidium Nosema ceranae on honey bee queen physiology (Apis mellifera)

Nosema ceranae, a microsporidian parasite originally described in the Asian honey bee Apis cerana, has recently been found to be cross-infective and to also parasitize the European honey bee Apis mellifera. Since this discovery, many studies have attempted to characterize the impact of this parasite in A. mellifera honey bees. Nosema species can infect all colony members, workers, drones and queens, but the pathological effects of this microsporidium has been mainly investigated in workers, despite the prime importance of the queen, who monopolizes the reproduction and regulates the cohesion of the society via pheromones. We therefore analyzed the impact of N. ceranae on queen physiology. We found that infection by N. ceranae did not affect the fat body content (an indicator of energy stores) but did alter the vitellogenin titer (an indicator of fertility and longevity), the total antioxidant capacity and the queen mandibular pheromones, which surprisingly were all significantly increased in Nosema-infected queens. Thus, such physiological changes may impact queen health, leading to changes in pheromone production, that could explain Nosema-induced supersedure (queen replacement).     

Caste-specific modulation of juvenile hormone III content and ecdysteroid titer in postembryonic development of the stingless bee,Scaptotrigona postica depilis

Summary Juvenile hormone III content and ecdysteroid titer were analyzed for larval and pupal development of the stingless bee,Scaptotrigona postica depilis. Castespecific differences in juvenile hormone III content were detected at three developmental phases: at the transition from the fourth to the fifth larval stadium, in the spinning phase of the fifth larval stadium, and shortly after the imaginal moult. During the fifth larval stadium, juvenile hormone content closely reflects corpora allata activity. Juvenile hormone synthesis may thus be responsible for the elevated hormone titer in spinning-phase queen larvae, a phase of known sensitivity for induction of queen characters by exogenous juvenile hormone. For ecdysteroids, two phases of caste-specific differences were found: in the pre-pupal phase, and shortly after the imaginal moult. In both periods the titer in queens is distinctly higher compared to workers.Abbreviations Im imago 1 day after eclosion - L3, L4, L5 larval instars 3, 4, and 5 - L5F1, L5F2 substages of feeding phase in fifth larval instar - L5S1, L5S2, L5S3 substages of spinning phase in fifth larval instar - PP1, PP2 substages of prepupal phase - Pw white eyed pupa - Pp pink eyed pupa - Pr red eyed pupa - Pd dark eyed pupa - Pdl, Pdm, Pdd dark eyed pupa with progressive tanning of cuticle - RIA radioimmunoassay     

Conflict over male parentage in stingless bees

Summary Stingless bees usually have one, singly-mated queen. This can lead to a genetic conflict of interest between the queen and the non-laying workers over who should produce the males. In many stingless bee species workers have developed ovaries and can produce male-destined eggs. In this study we compile the available data on who produces the males in stingless bees. Worker reproduction is common but less frequent than expected from predictions built on relatedness-based preferences of non-laying workers. We tested whether the pattern in worker reproduction can be explained best by queen control, by an arms race between workers and their queen, by the costs of losing workers to reproductive competition, or by phylogenetic constraints. The data are consistent with the view that there is ongoing conflict over male production that is resolved differently depending on the specific dynamics of costs and benefits of worker reproduction. There was also a role for phylogeny; Melipona workers often reproduced while Plebeia and Australian stingless bee workers seldom or never did. The high worker reproduction in Melipona may reflect low costs, because many of the replaced queen-laid eggs would become excess queens.Received 17 April 2003; revised 2 September 2003; accepted 5 September 2003.     

The juvenile hormone analog pyriproxyfen affects ecdysteroid-dependent cuticle melanization and shifts the pupal ecdysteroid peak in the honey bee (Apis mellifera)

The control of the pupal melanization in the honey bee by ecdysteroids, and the modulation of these processes by a juvenile hormone analog were investigated by a combination of in vivo and in vitro experiments. Injection of 1-5 microg of 20-hydroxyecdysone (20E) into unpigmented pupae showed a dose- and stage-dependent effect. The higher the dose and the later the injection was performed, the more pronounced was the delay in cuticle pigmentation. This inhibition of cuticular melanization by artificially elevated ecdysteroid titers was corroborated by in vitro experiments, culturing integument from unpigmented, dark-eyed pupae for 1-4 days in the presence of 20E (2 or 5 microg/ml culture medium). Topical application (1 microg) of pyriproxyfen to unpigmented, white-eyed pupae had the opposite effect, leading to precocious and enhanced melanization of the pupal cuticle. In vitro incubation of integuments in the presence of this juvenile hormone analog (1 microg/ml) confirmed these results, showing that pyriproxyfen is apparently capable of triggering melanization. The in vivo mode of action of pyriproxyfen was further investigated by quantifying hemolymph ecdysteroids by radioimmunoassays. Topical application leads to a delay of the pupal ecdysteroid peak by 4 days. The pyriproxyfen-induced low ecdysteroid titers during early pupal development could account for precocious pigmentation by removing an inhibition on prophenoloxidase activation normally imposed by the elevated ecdysteroid titer during this phase.     

Reproductive division of labor, dominance, and ecdysteroid levels in hemolymph and ovary of the bumble bee Bombus terrestris

To determine whether ecdysteroids are associated with reproductive division of labor in Bombus terrestris, we measured their levels in hemolymph and ovaries of queens and workers. Queens heading colonies had large active ovaries with high ecdysteroid content, whereas virgin gynes and mated queens before and after diapause had undeveloped ovaries with low ecdysteroid content. The hemolymph ecdysteroid titer was rather variable, but in a pooled analysis of mated queens before and after diapause versus colony-heading queens, ecdysteroid titers were higher in the latter group. In workers, agonistic behavior, ovarian activity, ovarian ecdysteroid content, and hemolymph ecdysteroid titers were positively correlated, and were lowest when a queen was present. In queenless workers, ecdysteroid levels were elevated in dominant workers, and were also influenced by the presence of brood and by group demography; hormone levels were higher in bees kept in larger groups. These findings are consistent with the premise that in B. terrestris the ovary is the primary site of ecdysteroid synthesis, and they show that ecdysteroids levels vary with the social environment.     

Ecdysteroid titer, ovary status, and dominance in adult worker and queen bumble bees (Bombus terrestris)

The role of ecdysteroids in the regulation of dominance and reproduction in social Hymenoptera is little explored. In the current study we compared ecdysteroid titers in hemolymph of individual queen and worker bumble bees (Bombus terrestris) that differ in their behavior, reproductive status and social environment. Egg-laying queens that head colonies and have ovaries exhibiting all stages of follicle development, had a higher ecdysteroid titer than virgin queens whose ovaries contained only follicles at initial stages. In workers, the relationship between ecdysteroid titers and follicle development appears to be more complex and to be influenced by the bee's social status and social environment. Shortly after emergence, young workers had only follicles at the initial stages of oogenesis and they exhibited a low ecdysteroid titer. No significant correlation was detected between ovary status and ecdysteroid titer in workers, with some workers showing activated ovaries but low ecdysteroid titers. However, at six days of age, a trend towards higher ecdysteroid titer was observed for workers in queenless groups, a condition characterized by rapid follicle development relative to queenright conditions. In these queenless groups, high social status was associated with high ecdysteroid titers. By contrast, in queenright workers ecdysteroid titers were low, even for bees with presumably high social status that had activated ovaries and were observed performing oviposition behavior. This study suggests that ecdysteroids are involved in regulation of reproduction in B. terrestris.     

Patriline differences in emergency queen rearing in the honey bee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

Summary In the polyandrous honey bee, Apis mellifera, workers can potentially increase their inclusive fitness by rearing full-sister queens. If the mother queen dies suddenly, workers feed a few larvae in worker cells with royal jelly and rear them into queens (emergency queen rearing). Using DNA microsatellite markers we determined the patriline of emergency queens reared in two colonies headed by naturally-mated queens before being made queenless. We found that some patrilines were reared more than others in one colony, but not in the other. These differences between colonies suggest that selective rearing is not always present and this might explain the mixed results of previous nepotism studies in the honey bee.Received 10 February 2003; revised 7 March 2003; accepted 17 March 2003.     

Molecular characterization of the gene feminizer in the stingless bee Melipona interrupta (Hymenoptera: Apidae) reveals association to sex and caste development

In highly eusocial insects, development of reproductive traits are regulated not only by sex determination pathway, but it also depends on caste fate. The molecular basis of both mechanisms in stingless bees and possible interaction with each other is still obscure. Here, we investigate sex determination in Melipona interrupta, focusing on characterization and expression analysis of the feminizer gene (Mi-fem), and its association to a major component of caste determination, the juvenile hormone (JH). We present evidence that Mi-fem mRNA is sex-specifically spliced in which only the female splice variant encodes the full length protein, following the same principle known for other bee species. We quantified Mi-fem expression among developmental stages, sexes and castes. Mi-fem expression varies considerably throughout development, with higher expression levels in embryos. Also, fem levels in pupae and newly emerged adults were significantly higher in queens than workers and males. Finally, we ectopically applied JH in cocoon spinning larvae, which correspond to the time window where queen/worker phenotypes diverge. We observed a significantly increase in Mi-fem expression compared to control groups. Since up to 100% of females turn into queens when treated with JH (while control groups are composed mainly of workers), we propose that fem might act to regulate queens' development. Our findings provide support for the conserved regulatory function of fem in Melipona bees and demonstrate a significant correlation between key elements of sex and caste determination pathways, opening the avenue to further investigate the molecular basis of these complex traits.     

Non-destructive sources of DNA used to genotype honey bee (Apis mellifera) queens

We describe a method for genotyping honey bee queens Apis mellifera L. (Hymenoptera: Apidae), using biological materials that are normally cast off during development (larval and pupal exuviae), or can be removed without apparent damage to queen longevity or acceptability to workers (wing clippings). Highly polymorphic microsatellite loci were successfully amplified from DNA from all of these sources, although with differing degrees of success. DNA was extracted using a simple Chelex 100® boiling procedure. Four microsatellite primers were used to amplify the DNA, and the PCR products were visualized on an ALFexpress Automated Sequencer. Genotypes created from these sources were consistent with those originating from tarsal tissue. Successful retrieval and amplification of DNA from the exuviae from immature queens allows potential breeding individuals to be genotyped and selected before they become adults. This procedure may therefore have value as DNA marker‐assisted breeding programs are developed for honey bees.     

Deformed wing virus in western honey bees (Apis mellifera) from Atlantic Canada and the first description of an overtly-infected emerging queen

Deformed wing virus (DWV) in western honey bees (Apis mellifera) often remains asymptomatic in workers and drones, and symptoms have never been described from queens. However, intense infections linked to parasitism by the mite Varroa destructor can cause worker wing deformity and death within 67 h of emergence. Ten workers (eight with deformed wings and two with normal wings) and three drones (two with deformed wings and one with normal wings) from two colonies infected with V. destructor from Nova Scotia, Canada, and two newly-emerged queens (one with deformed wings and one with normal wings) from two colonies infected with V. destructor from Prince Edward Island, Canada, were genetically analyzed for DWV. We detected DWV in all workers and drones, regardless of wing morphology, but only in the deformed-winged queen. This is the first report of DWV from Atlantic Canada and the first detection of a symptomatic queen with DWV from anywhere.     

Absence of consistent diel rhythmicity in mated honey bee queen behavior

Relatively little is known about the temporal control of behavior of honey bee queens under natural conditions. To determine if mated honey bee queens possess diel rhythmicity in behavior, we observed them in glass-sided observation hives, employing two focal studies involving continuous observations of individual queens as well as a scan-sampling study of multiple queens. In all cases, all behaviors were observed at all times of the day and night. In four of the five queens examined in focal studies, there were no consistent occurrences of diel periodicity for any of the individual behaviors. A more encompassing measure for periodicity, in which the behaviors were characterized as active (walking, inspecting, egg-laying, begging for food, feeding, and grooming self) or inactive (standing), also failed to reveal consistent diel rhythmicity. Furthermore, there were no consistent diel differences in the number of workers in the queen's retinue. Behavioral arrhythmicity persisted across seasons and despite daily changes in both light and temperature levels. Both day and night levels of behavioral activity were correlated with daytime, but not with nighttime, ambient temperatures. The behavior of the one exceptional queen was not consistent: diurnal activity patterns were present during two 24-h observation sessions but arrhythmicity during another. Based on the behavior observed by all but one of the queens examined in this work, the arrhythmic behavior by the mated honey bee queen inside the colony appears to be similar to that exhibited by worker bees before they approach the age of onset of foraging behavior.     

Detection of multiple viruses in queens of the honey bee Apis mellifera L

Individual honey bee Apis mellifera L. queens were examined for the presence of six honey bee viruses including acute bee paralysis virus, chronic bee paralysis virus, black queen cell virus, deformed wing virus, Kashmir bee virus, and sacbrood virus. All viruses, except ABPV, were detected in the samples. Among queens examined for virus infections, 93% had multiple virus infections. The detection of viruses in queens raises the possibility of a vertical transmission pathway wherein infected queens can pass virus through their eggs to their offspring.     

Larval juvenile hormone treatment affects pre-adult development,but not adult age at onset of foraging in worker honey bees (Apis mellifera)

Previous research has shown that juvenile hormone (JH) titers increase as adult worker honey bees age and treatments with JH, JH analogs and JH mimics induce precocious foraging. Larvae from genotypes exhibiting faster adult behavioral development had significantly higher levels of juvenile hormone during the 2nd and 3rd larval instar. It is known that highly increased JH during this period causes the totipotent female larvae to differentiate into a queen. We treated third instar larvae with JH to test the hypothesis that this time period may be a developmental critical period for organizational effects of JH on brain and behavior also in the worker caste, such that JH treatment at a lower level than required to produce queens will speed adult behavioral development in workers. Larval JH treatment did not influence adult worker behavioral development. However, it made pre-adult development more queen-like in two ways: treated larvae were capped sooner by adult bees, and emerged from pupation earlier. These results suggest that some aspects of honey bee behavioral development may be relatively insensitive to pre-adult perturbation. These results also suggest JH titer may be connected to cues perceived by the adult bees indicating larval readiness for pupation resulting in adult bee cell capping behavior.     

Caste-specific differences in ecdysteroid titers in early larval stages of the bumblebee Bombus terrestris

Mounting evidence implicates ecdysteroids in queen-worker differentiation during the last larval instars of highly social insects. In the present study, we analyzed ecdysteroid titers in queen and worker larvae of the bumblebee Bombus terrestris from the second to the early fourth instar. B. terrestris is of particular interest because caste is already determined in the second instar, presumably by a pheromonal signal emitted by the egg-laying queen. Caste differences in the adults, however, are only expressed at the physiological and not at the morphological level, except for the distinctly larger size of the queen. In the second and third instar, ecdysteroid titers in queen larvae were generally higher than those of workers. These early caste-specific differences, however, were abolished in the fourth instar. In the early fourth instar we could detect two small ecdysteroid peaks, with the one preceding the cocoon-spinning phase presenting the characteristics of a pupal commitment peak. The synchrony of caste differences in ecdysteroid and juvenile hormone titers suggests a synergistic action of these hormones in caste determination.