Effect of honey bee queen mating condition on worker ovary activation

The presence of the honey bee queen reduces worker ovary activation. When the queen is healthy and fecund, this is interpreted as an adaptive response as workers can gain fitness from helping the queen raise additional offspring, their sisters. However, when the queen is absent, workers activate their ovaries and lay unfertilized eggs that become males. Queen pheromones are recognised as a factor affecting worker ovary activation. Recent work has shown that queen mandibular pheromone composition changes with queen mating condition and workers show different behavioural responses to pheromone extracts from these queens. Here, we tested whether workers reared in colonies with queens of different mating condition varied in level of ovary activation. We also examined the changes in the chemical composition of the queen mandibular glands to determine if the pheromone blend varied among the queens. We found that the workers activated their ovaries when queens were unmated and had lower ovary activation when raised with mated queens, suggesting that workers detect and respond adaptively to queens of differing mating status. Moreover, variation in queen mandibular gland’s chemical composition correlated with the levels of worker ovary activation. Although correlative, this evidence suggests that queen pheromone may act as a signal of queen mating condition for workers, in response to which they alter their level of ovary activation.     

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.     

Juvenile hormone effect on DNA synthesis and apoptosis in caste-specific differentiation of the larval honey bee (Apis mellifera L.) ovary

Caste-specific differentiation of the honey bee ovary commences in the last larval instar. In this process, formation of germ cell clusters by synchronous and incomplete mitoses occurs in the queen ovary, whereas in the worker ovary programmed cell death is the dominant feature. BrdU and TUNEL labeling were used to study dynamics of cell proliferation and apoptosis-dependent DNA degradation in ovaries of naturally developing queens and workers, as well as in juvenile hormone-treated worker larvae. Cell proliferation in ovaries of last-instar queen larvae generally exceeded that in workers, except for the late feeding phase. This inversion in cell proliferation patterns coincided with the onset of apoptosis in worker ovaries, as evidenced by TUNEL labeling. Juvenile hormone application to early-fifth-instar worker larvae had two noticeable effects. First, it diminished the number of S-phase nuclei in ovaries of late feeding-phase workers, bringing them to queen-like levels. Second, it prevented the induction of apoptotic DNA degradation. Caste-specific regulation of cell division in connection with programmed cell death can thus be attributed to the previously described differences in juvenile hormone titer in queen and worker larvae, adding a new facet to this hormone's multiple functions.     

Queen-signal modulation of worker pheromonal composition in honeybees

Worker sterility in honeybees is neither absolute nor irreversible. Whether under queen or worker control, it is likely to be mediated by pheromones. Queen-specific pheromones are not exclusive to queens; workers with activated ovaries also produce them. The association between ovarian activation and queen-like pheromone occurrence suggests the latter as providing a reliable signal of reproductive ability. In this study we investigated the effect of queen pheromones on ovary development and occurrence of queen-like esters in workers' Dufour's gland. Workers separated from the queenright compartment by a double mesh behaved like queenless workers, activating their ovaries and expressing a queen-like Dufour's gland secretion, confirming that the pheromones regulating both systems are non-volatile. Workers with developed ovaries produced significantly more secretion than sterile workers, which we attribute primarily to increased ester production. Workers separated from the queenright compartment by a single mesh displayed a delayed ovarian development, which we attribute to interrupted transfer of the non-volatile pheromone between compartments. We suggest that worker expression of queen-like characters reflects a queen-worker arms race; and that Dufour's gland secretion may provide a reliable signal for ovarian activation. The associative nature between ovary development and Dufour's gland ester production remains elusive.     

Honey bee queen mandibular pheromone inhibits ovary development and fecundity in a fruit fly

A key feature of eusocial insects is their reproductive division of labour. The queen signals her fecundity to her potentially reproductive daughters via a pheromone, which renders them sterile. In contrast, solitary insects lack division in reproductive labour and there is no such social signalling or need for ovary‐regulating pheromones. Nonetheless, females from both non‐social and eusocial lineages are expected to regulate their ovaries to maximize inclusive lifetime reproductive success. It is not known, however, whether the underlying networks that regulate ovary activation are homologous between non‐social and eusocial taxa, especially when these taxa are phylogenetically distant. In this study, we provide evidence that solitary fruit flies may share a conserved ovary‐regulating pathway with a eusocial honey bee, Apis mellifera L. (Hymenoptera: Apidae). Specifically, we demonstrate that honey bee queen mandibular pheromone (QMP) inhibits fly ovaries in much the same way as it suppresses worker ovaries. Drosophila melanogaster Meigen (Diptera: Drosophilidae) exposed to sufficient doses of QMP showed a reduction in ovary size, produced fewer eggs, and generated fewer viable offspring, relative to unexposed controls. Drosophila melanogaster therefore responds to an interspecific social cue to which it would not normally be exposed. Although we cannot strictly rule out an incidental effect, this conspicuous response suggests that these two species may share an underlying mechanism for ovary regulation. Why a non‐social species of fly responds to a highly social bee's pheromone is not clear, but one possibility is that solitary and social insects share pathways associated with female reproduction, as predicted by the ‘groundplan’ hypothesis of social evolution.     

Worker laying in leafcutter ant Acromyrmex subterraneus brunneus （Formicidae, Attini）

We studied the process of offspring production in queenless colonies of Acromyrmex subterraneus brunneus, and particularly evaluated the ovary development of workers as a function of their age. For this, subcolonies were set up and evaluated at different periods of isolation from the queen （2, 4 and 6 months）, besides individually labeled age groups. The subcolonies were assessed according to offspring production and ovaries containing oocytes or not. The evaluations showed worker oviposition and development of males originating from worker-laid eggs. At 2 months＇ absence of the queen, eggs and larvae were found, with eggs in a higher proportion than larvae. After 4 months, the proportion of eggs had reduced while larvae had increased, and a pupa was found in one subcolony. At 6 months, besides a higher share of larvae, one pupa and one adult male were found. Dissection of workers revealed ovaries containing oocytes during the periods of evaluation. Only a group of medium-sized and large workers, 23.3%, 20.9% and 37.5% of the population from each period assessed in queenless subcolonies respectively, presented developed oocytes in the ovary. The same was observed in colonies with a queen, with 17.6%, 19.6% and 7.8% of the group of dissected workers from each time period, respectively. With respect to worker age, we observed by dissection of the ovary, that the greatest percentage of individuals with ovarioles containing oocytes occurred at 45 days （6 weeks） up to 90 days （12 weeks）. These results probably are associated with the workers reproduction and the laying of trophic and reproductive eggs in colonies with and without a queen; these eggs have distinct functions in each situation.     

Queen pheromones affecting the production of queen-like secretion in workers

The honeybee queen pheromones promote both worker sterility and worker-like pheromone composition; in their absence workers become fertile and express the queen pheromones. Which of the queen pheromones regulate worker pheromone expression and how, is still elusive. Here we investigated how two queen pheromones, the mandibular and Dufour’s, singly or combined, affect worker ovarian activation and occurrence of queen-like Dufour’s esters. Although queen mandibular pheromone (QMP) alone, or combined with Dufour’s secretion, inhibited to some extent worker reproduction, neither was as effective as the queen. The effect of the queen pheromones on worker pheromone expression was limited to workers with developed ovaries. Here too, QMP and Dufour’s combined had the greatest inhibitory effect. In contrast, treatment with Dufour’s alone resulted in augmentation of esters in the workers. This is another demonstration that a pheromone emitted by one individual affects the rates of its production in another individual. Ester production was tightly coupled to ovarian development. However fertile workers from queenright or QMP-treated colonies had significantly higher amounts of esters in their Dufour’s gland than untreated queenless colonies. The fact that the queen or QMP exert greater suppression on signal production than on ovary activation, suggests disparate regulatory pathways, and presents a challenging ultimate as well as proximate questions.     

Age and Behavior of Honey Bee Workers,Apis mellifera,that Interact with Drones

Colony reproduction in honey bees involves complex interactions between sterile workers and reproductive castes. Although worker–queen interactions have been studied in detail, worker–drone interactions are less well understood. We investigated caste interactions in honey bees by determining the age and behavior of workers that perform vibration signals, trophallaxis, and grooming with drones. Workers of all ages could engage in the different interactions monitored, although workers that performed vibration signals on drones were significantly older than those engaging in trophallaxis and grooming. Only 3–8% of workers engaged in the different behaviors were monitored. Compared with workers that performed vibration signals only on workers (‘worker vibrators’), those that performed signals on both workers and drones (‘drone vibrators’) had greater movement rates inside the nest, higher vibration signaling rates, and were more likely to have an immediate association with foraging. Both worker vibrators and drone vibrators contacted drones of all ages as they moved through the nest. However, drone vibrators contacted drones at higher rates, contacted slightly, but significantly younger drones, and were more likely to engage in trophallaxis and grooming with drones, in addition to vibrating them. Taken together, our results suggest that tiny proportions of workers belonging to separate, but overlapping age groups provide most of the care received by adult drones, and that drone vibrators comprise a subset of signalers within a colony that have an increased tendency to contact and interact with drones. Vibratory, tactile signals are involved in colony reproductive and movement decisions in a number of species of bees, wasps and ants, and may provide valuable tools for investigating caste interactions in many insect societies.     

Factors affecting ovary activation in honey bee workers: a meta-analysis

A conspicuous feature of honey bee social biology is the division of labour between reproductive queens and functionally sterile workers. However, the sterility of workers is conditional and sensitive to genetic and environmental context. Despite this understanding, we do not yet know how effective differences in genotype versus differences in colony environment are for generating variation in levels of ovary activation in a population of workers. We therefore performed a field study and meta-analysis to estimate the standardized effect size g of broad ‘environmental’ and ‘genetic’ manipulations on worker ovary scores. Despite considerable differences in methodology and treatment among published studies, we report that both genetic and environmental manipulations were effective at generating differences in ovary phenotype between groups of worker bees. Our analysis found that environmental treatments, such as differences in pheromones and diet, had a larger mean effect on worker ovary activation scores than have genetic factors such as patriline or strain (g = 0.54 vs. 0.39). We conclude by discussing the biological significance of environmentally sensitive sterility in honey bee societies.     

Sequence and Expression Characteristics of Long Noncoding RNAs in Honey Bee Caste Development – Potential Novel Regulators for Transgressive Ovary Size

Division of labor in social insect colonies relies on a strong reproductive bias that favors queens. Although the ecological and evolutionary success attained through caste systems is well sketched out in terms of ultimate causes, the molecular and cellular underpinnings driving the development of caste phenotypes are still far from understood. Recent genomics approaches on honey bee developmental biology revealed a set of genes that are differentially expressed genes in larval ovaries and associated with transgressive ovary size in queens and massive cell death in workers. Amongst these, two contigs called special attention, both being over 200 bp in size and lacking apparent coding potential. Herein, we obtained their full cDNA sequences. These and their secondary structure characteristics placed in evidence that they are bona fide long noncoding RNAs (lncRNA) differentially expressed in larval ovaries, thus named lncov1 and lncov2. Genomically, both map within a previously identified QTL on chromosome 11, associated with transgressive ovary size in honey bee workers. As lncov1 was over-expressed in worker ovaries we focused on this gene. Real-time qPCR analysis on larval worker ovaries evidenced an expression peak coinciding with the onset of autophagic cell death. Cellular localization analysis through fluorescence in situ hybridization revealed perinuclear spots resembling omega speckles known to regulate trafficking of RNA-binding proteins. With only four lncRNAs known so far in honey bees, two expressed in the ovaries, these findings open a novel perspective on regulatory factors acting in the fine tuning of developmental processes underlying phenotypic plasticity related to social life histories.     

Workers do not mediate the inhibitory power of queens in a termite, <Emphasis Type="Italic">Reticulitermes speratus</Emphasis> (Isoptera,Rhinotermitidae)

In social insects, the caste systems are based on reproductive division of labor; queens specialize in reproduction and workers primarily maintain the colony. Recently, a volatile pheromone containing n-butyl-n-butyrate and 2-methyl-1-butanol was identified as a termite queen pheromone that inhibits the differentiation of female neotenic reproductives (secondary queens). Although this volatile inhibitory pheromone regulates caste differentiation directly, the method by which it reaches members without direct contact with the queen in large colonies is not well understood. Therefore, additional mechanisms of indirect communication must exist, such as worker-mediated queen signal transport. We found that workers exposed to female reproductives did not mediate queens’ inhibitory signal in a termite Reticulitermes speratus. The experiment assessed worker transfer from direct to indirect contact groups and determined that the differentiation of new female reproductives in the indirect contact groups was not influenced by the direct contact groups, whereas direct contact with functional female reproductives and artificial queen pheromone did suppress neotenic differentiation. This suggests that worker transfer of the queen signal is unlikely and that for colony-wide inhibition direct contact by the majority of infertile members with reproductives or eggs, which emit the same volatiles as female reproductives, is necessary within a certain time interval.     

The effects of honey bee (Apis mellifera L.) queen reproductive potential on colony growth

Reproduction in species of eusocial insects is monopolized by one or a few individuals, while the remaining colony tasks are performed by the worker caste. This reproductive division of labor is exemplified by honey bees (Apis mellifera L.), in which a single, polyandrous queen is the sole colony member that lays fertilized eggs. Previous work has revealed that the developmental fate of honey bee queens is highly plastic, with queens raised from younger worker larvae exhibiting higher measures in several aspects of reproductive potential compared to queens raised from older worker larvae. Here, we investigated the effects of queen reproductive potential (“quality”) on the growth and winter survival of newly established honey bee colonies. We did so by comparing the growth of colonies headed by “high-quality” queens (i.e., those raised from young worker larvae, which are more queen-like morphologically) to those headed by “low-quality” queens (i.e., those raised from older worker larvae, which are more worker-like morphologically). We confirmed that queens reared from young worker larvae were significantly larger in size than queens reared from old worker larvae. We also found a significant positive effect of queen grafting age on a colony’s production of worker comb, drone comb, and stored food (honey and pollen), although we did not find a statistically significant difference in the production of worker and drone brood, worker population, and colony weight. Our results provide evidence that in honey bees, queen developmental plasticity influences several important measures of colony fitness. Thus, the present study supports the idea that a honey bee colony can be viewed (at least in part) as the expanded phenotype of its queen, and thus selection acting predominantly at the colony level can be congruent with that at the individual level.     

Social Modulation of Stress Reactivity and Learning in Young Worker Honey Bees

Alarm pheromone and its major component isopentylacetate induce stress-like responses in forager honey bees, impairing their ability to associate odors with a food reward. We investigated whether isopentylacetate exposure decreases appetitive learning also in young worker bees. While isopentylacetate-induced learning deficits were observed in guards and foragers collected from a queen-right colony, learning impairments resulting from exposure to this pheromone could not be detected in bees cleaning cells. As cell cleaners are generally among the youngest workers in the colony, effects of isopentylacetate on learning behavior were examined further using bees of known age. Adult workers were maintained under laboratory conditions from the time of adult emergence. Fifty percent of the bees were exposed to queen mandibular pheromone during this period, whereas control bees were not exposed to this pheromone. Isopentylacetate-induced learning impairments were apparent in young (less than one week old) controls, but not in bees of the same age exposed to queen mandibular pheromone. This study reveals young worker bees can exhibit a stress-like response to alarm pheromone, but isopentylacetate-induced learning impairments in young bees are suppressed by queen mandibular pheromone. While isopentylacetate exposure reduced responses during associative learning (acquisition), it did not affect one-hour memory retrieval.     

Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees

The mandibular glands of queen honeybees produce a pheromone that modulates many aspects of worker honeybee physiology and behavior and is critical for colony social organization. The exact chemical blend produced by the queen differs between virgin and mated, laying queens. Here, we investigate the role of mating and reproductive state on queen pheromone production and worker responses. Virgin queens, naturally mated queens, and queens instrumentally inseminated with either semen or saline were collected 2 days after mating or insemination. Naturally mated queens had the most activated ovaries and the most distinct chemical profile in their mandibular glands. Instrumentally inseminated queens were intermediate between virgins and naturally mated queens for both ovary activation and chemical profiles. There were no significant differences between semen- and saline-inseminated queens. Workers were preferentially attracted to the mandibular gland extracts from queens with significantly more activated ovaries. These studies suggest that the queen pheromone blend is modulated by the reproductive status of the queens, and workers can detect these subtle differences and are more responsive to queens with higher reproductive potential. Furthermore, it appears as if insemination substance does not strongly affect physiological characteristics of honeybee queens 2 days after insemination, suggesting that the insemination process or volume is responsible for stimulating these early postmating changes in honeybee queens.     

Potential and realized reproduction by different worker castes in queen-less and queen-right colonies of <Emphasis Type="Italic">Pogonomyrmex badius</Emphasis>

Workers of the Florida harvester ant (Pogonomyrmex badius), the only North American Pogonomyrmex with a polymorphic worker caste, produce males when colonies are orphaned. In this study,we assessed the reproductive potential of workers of each caste group, minors and majors, in the presence and absence of the queen, and tested whether males produced in natural queen-right colonies are derived from workers. Worker size was positively correlated with ovariole number such that major workers had approximately double the number of ovarioles as minor workers. The number of vitellogenic oocytes, a measure of reproductive potential, was greater in major compared to minor workers and increased in both worker castes when queens were removed. Major workers have greater reproductive potential than minors although they represent a minority within the colony (~5% of workers are majors). Worker produced eggs were visible in colonies 28 – 35 days after queen removal. This time lag, from queen removal to egg production, is similar to other ants and bees. Though workers are capable of producing viable eggs, we found no evidence that they do so in queen-right colonies, suggesting that worker reproduction is controlled via some social mechanism (self restraint, policing, or inhibition). This result supports predictions of kin selection theory – that due to multiple mating by the queen workers are more related to queen-produced males than most worker-produced males and should thus favor reproduction by the queen and inhibit reproduction by other workers. Received 25 January 2007; revised 1 May 2007; accepted 21 May 2007.     

Juvenile-hormone-dependent interaction of actin and spectrin is crucial for polymorphic differentiation of the larval honey bee ovary

Programmed cell death in the worker ovary of Apis mellifera reduces the number of ovarioles during metamorphosis from 150-200 primordia to less than 10. In contrast, practically all ovarioles in the ovary of queens survive to the adult stage. The correct formation and persistence of polyfusomes has been suggested as a critical factor for ovariole survival. We have analyzed the developmental dynamics of F-actin and alpha-spectrin in fusomes of queen and worker larvae, and in juvenile-hormone-treated worker larvae. Small fusomes containing actin and spectrin can be detected in the ovaries of fourth instar larvae in both castes. After molting to the fifth instar, the actin-spectrin association persists in the enlarged fusomes of queen ovarioles. In workers, actin dissociates from the fusomal and cortical alpha-spectrin. Coinciding with the appearance of apoptosis markers, large agglomerates of actin are detectable in worker ovarioles. Treatment of fourth-instar worker larvae with juvenile hormone rescues ovarioles from apoptosis and maintains the actin-spectrin association. Juvenile-hormone-dependent actin-spectrin interaction is thus one of the earliest steps in the differentiation of a polymorphic ovary. Plasticity in ovariole numbers as a result of hormone-dependent fusome formation may be a more widespread phenomenon in insects, extending beyond caste polymorphism in highly eusocial Hymenoptera.     

Durability of information concerning the presence of a gamergate in Diacamma sp. from Japan

In social insects, information on the presence of a queen is known to affect the reproductive behaviour of workers. In the queenless ant Diacamma sp., information of the presence of the gamergate, the functional queen, is transmitted exclusively by direct physical contact between the gamergate and workers. Periodic contacts between the gamergate and each worker are therefore necessary to maintain worker sterility. However, how the difference in the contact interval influences reproductive behaviour of workers is unclear. In the present study, by artificially controlling the exact interval of physical contact between workers with the gamergate, it is shown that the inter‐contact interval influences the worker's reproductive condition (i.e. a longer interval leads to more developed ovaries). This system provides a good opportunity to study the mechanisms underling the colony size dependence of individual as well as colony characteristics.