Queenright Workers Research Articles

Differential gene expression underlying the biosynthesis of Dufour's gland signals in Bombus impatiens.

Pheromones regulating social behavior are one of the most explored phenomena in social insects. However, compound identity, biosynthesis and their genetic basis are known in only a handful of species. Here we examined the gene expression associated with pheromone biosynthesis of two main chemical classes: esters and terpenes, using the social bee Bombus impatiens. We conducted chemical and RNA-seq analyses of the Dufour's gland, an exocrine gland producing a plethora of pheromones regulating social behavior in hymenopteran species. The Dufour's gland contains mostly long-chained hydrocarbons, terpenes and esters that signal reproductive and social status in several bee species. In bumble bees, the Dufour's gland contains queen- and worker-specific esters, in addition to terpenes and terpene-esters only found in gynes and queens. These compounds are assumed to be synthesized de novo in the gland, however, their genetic basis is unknown. A whole transcriptome gene expression analysis of the gland in queens, gynes, queenless and queenright workers showed distinct transcriptomic profiles, with thousands of differentially expressed genes between the groups. Workers and queens express genes associated with key enzymes in the biosynthesis of wax esters, while queens and gynes preferentially express key genes in terpene biosynthesis. Overall, our data demonstrate gland-specific regulation of chemical signals associated with social behavior and identifies candidate genes and pathways regulating caste-specific chemical signals in social insects.

Investigating queen influence on worker behaviour using comparisons of queenless and queenright workers

Female eusocial sweat bees are capable of behaving as queens or workers. Relatively few females become queens, and those that do can directly manipulate the reproductive behaviour of other females in the nest. We collected Lasioglossum (Dialictus) laevissimum workers from nests with and without queens (queenright and queenless nests, respectively) to investigate the influence queens exert on worker behaviour via direct manipulation. Overall, very few L. laevissimum workers (17%) had developed ovaries in Ontario, but queenright and queenless workers were equally likely to have developed ovaries and worn mandibles. However, queenless workers were more likely to be mated than queenright workers. These results suggest first, that queens inhibit egg-laying in most, but not all workers, and second, that queen behaviour during the first few days of workers’ adult lives exerts a lasting influence on worker behaviour. We also compared social traits of L. laevissimum and other Dialictus species using principal components analysis. A strong correlation between worker reproduction and male availability suggests that queen manipulation of the worker brood sex ratio has evolved as an indirect mechanism for queens to discourage worker reproduction.

Comparative transcriptome analysis on the synthesis pathway of honey bee (Apis mellifera) mandibular gland secretions

Secretions from mandibular glands (MGs) have important caste-specific functions that are associated with the social evolution of honey bees. To gain insights into the molecular architecture underlying these caste differences, we compared the gene expression patterns of MGs from queens, queenright workers (WQRs) and queenless workers (WQLs) using high-throughput RNA-sequencing technology. In total, we identified 46 candidate genes associated with caste-specific biosynthesis of fatty acid pheromones in the MG, including members of cytochrome P450 (CYP450) family and genes involved in fatty acid β-oxidation and ω-oxidation. For further identification of the CYP450s genes involved in the biosynthesis of MG secretions, we analyzed by means of qPCR, the expression levels of six of the CYP450 genes most abundantly expressed in the transcriptome analysis across different castes, ages, tasks and tissues. Our analysis revealed that CYP6AS8 and CYP6AS11, the most abundantly expressed CYP450 genes in worker and queen MGs, respectively, are selectively expressed in the MGs of workers and queens compared to other tissues. These results suggest that these genes might be responsible for the critical bifurcated hydroxylation process in the biosynthesis pathway. Our study contributes to the description of the molecular basis for the biosynthesis of fatty acid-derived pheromones in the MGs.

Physiology of reproductive worker honey bees (Apis mellifera): insights for the development of the worker caste.

Reproductive and behavioural specialisations characterise advanced social insect societies. Typically, the honey bee (Apis mellifera) shows a pronounced reproductive division of labour between worker and queen castes, and a clear division of colony roles among workers. In a queenless condition, however, both of these aspects of social organisation break down. Queenless workers reproduce, forage and maintain their colony operating in a manner similar to communal bees, rather than as an advanced eusocial group. This plasticity in social organisation provides a natural experiment for exploring physiological mechanisms of division of labour. We measured brain biogenic amine (BA) levels and abdominal fat body vitellogenin gene expression levels of workers in queenright and queenless colonies. Age, ovary activation and social environment influenced brain BA levels in honey bees. BA levels were most influenced by ovary activation state in queenless bees. Vitellogenin expression levels were higher in queenless workers than queenright workers, but in both colony environments vitellogenin expression was lower in foragers than non-foragers. We propose this plasticity in the interacting signalling systems that influence both reproductive and behavioural development allows queenless workers to deviate significantly from the typical worker bee reaction norm and develop as reproductively active behavioural generalists.

Associations between reproduction and work in workers of the Asian hive bee Apis cerana

If a honey bee (Apis spp.) colony becomes queenless, about 1/3 of young workers activate their ovaries and produce haploid male-producing eggs. In doing so queenless workers maximize their inclusive fitness because the normal option of vicarious production of relatives via their queen’s eggs is no longer available. But if many workers are engaged in reproduction, how does a queenless colony continue to feed its brood and forage? Here we show that in the Asian hive bee Apis cerana hypopharyngeal gland (HPG) size is larger in queenless workers than in queenright workers and that bees undertaking brood-rearing tasks have larger HPG than same-aged bees that are foraging. In queenless colonies, workers with a smaller number of ovarioles are more likely to have activated ovaries. This reinforces the puzzling observation that a large number of ovarioles reduces reproductive success in queenless A. cerana. It further suggests that reproductive workers either avoid foraging or transition to foraging later in life than non-reproductive workers. Finally, our study also showed that ovary activation and larger-than-average numbers of ovarioles had no statistically detectable influence on foraging specialization for pollen or nectar.

Worker reproductive parasitism in naturally orphaned colonies of the Asian red dwarf honey bee, Apis florea

The truce between honey bee (Apis spp.) workers over reproduction is broken in the absence of their queen. Queenright workers generally abstain from personal reproduction, raising only the queen’s offspring. Queenless workers activate their ovaries, produce eggs, and reduce the rate at which they destroy worker-laid eggs, so that some eggs are reared to maturity. Reduced policing of worker-laid eggs renders queenless nests vulnerable to worker reproductive parasitism (WRP), and may result in the colony raising eggs of unrelated (non-natal) workers that parasitize it. Queenless colonies of A. florea are heavily parasitized with the eggs of non-natal workers. However, queenless colonies often abscond upon disturbance and build a small comb in which to rear their own male offspring. We investigated three naturally occurring orphaned colonies to determine if they are also parasitized. We show that WRP is present in orphaned colonies, and non-natal workers have significantly higher rates of ovary activation than natal workers. In contrast to experimentally manipulated colonies, in our samples, natal and non-natal workers had statistically equal reproductive success, but this may have been due to the small number of non-natals present.

Worker reproductive parasitism and drift in the western honeybee Apis mellifera

When a honeybee (Apis spp.) colony loses its queen and is unable to rear a new one, some of the workers activate their ovaries and produce eggs. When a colony has a queen (i.e., it is queenright) almost all worker-laid eggs are eaten, but when hopelessly queenless, the workers become more tolerant of worker-laid eggs and rear some of them to adult drones. This increased tolerance renders a queenless colony vulnerable to worker reproductive parasitism, wherein unrelated workers enter the colony and lay eggs. Here, we show that the proportion of unrelated (non-natal) workers significantly decreases after an Apis mellifera colony becomes queenless. The remaining non-natal workers are as likely to have activated ovaries as natal workers, yet they produce more eggs than natal workers, resulting in significantly higher reproductive success for non-natal workers. In a second experiment, we provided queenless and queenright workers with a choice to remain in their own colony or to join a queenless or queenright colony nearby. The experiment was set up such that worker movement was unlikely to be due to simple orientation errors. Very few workers joined another colony, and there was no preference for workers to drift into or out of queenless or queenright colonies, in accordance with the proportion of non-natal workers declining significantly after becoming queenless in the first experiment.

The gene road to royalty – differential expression of hydroxylating genes in the mandibular glands of the honeybee

The advances in honeybee sociogenomics have paved the way for the study of social communication processes at the gene level, in particular the expression of caste-specific pheromones. The queen honeybee mandibular pheromone provides an excellent model system, in that biosynthesis of the hydroxylating fatty acid caste-specific pheromone appears to be reduced to a single chemical hydroxylation step of stearic acid. Queens are typified by omega-1-hydroxylation, as opposed to the worker-typical omega-hydroxylation. We hypothesized that this bifurcation is the consequence of differential expression of caste-specific genes that code for fatty acid-hydroxylating enzymes from the cytochrome P450 (CYP) family. Bioinformatics studies disclosed two candidate proteins CYP4AA1 and CYP18A1. We thus investigated the expression of these genes in the mandibular glands of queens, and of queenright (QR) and queenless (QL) workers. The real-time PCR results revealed that CYP4AA1 (omega-hydroxylation) was expressed at high levels in both QR and QL workers, whereas in queens its expression was negligible. The expression of CYP18A1 (omega-1-hydroxylation), on the other hand, was high in the queen's glands and negligible in those of QR workers. In QL workers, however, the expression of CYP18A1 was considerably elevated and significantly greater than in QR workers. Three-dimensional structural models constructed for these enzymes demonstrate differences in the active site between CYP18A1 and CYP4AA1, in line with their differential catalytic specificity. The fact that queen pheromone plasticity can be tracked all the way to gene expression provides a new insight into the process of caste differentiation and the accompanying social communication.

Regulation of hypopharyngeal gland activity and oogenesis in honey bee ( Apis mellifera) workers

In the honey bee, vitellogenin has several functions in addition to egg provisioning. Among others, it serves as a precursor to brood food proteins secreted by the hypopharyngeal glands of worker bees. In queenless workers with developing gonads, oogenesis and development of the hypopharyngeal glands are correlated. Here we describe two experiments that explored whether this relationship also exists in non-reproductive workers, and investigated a possible role of ecdysteroid hormones in the regulation of vitellogenin uptake. In the first experiment, the correlation between oocyte length and hypopharyngeal gland development was measured in workers before and after de-queening. In the second experiment, we induced middle-aged bees with resting glands to suddenly initiate brood care behaviour, and measured haemolymph ecdysteroid and vitellogenin titres. A strong positive relationship existed between morphometrical parameters of hypopharyngeal glands and ovaries in both queenless and queenright (functionally sterile) workers. No response of ecdysteroid titres to the addition of brood was detected in experiment 2, but high concentrations were measured in a small group of bees characterised by the possession of oocytes on the brink of yolk incorporation. We conclude that hypopharyngeal glands may belong to a previously described group of reproduction-related traits that are pleiotropically regulated in workers. A possible role for ecdysteroids in honey bee reproduction is discussed.

Brain modulation of Dufour’s gland ester biosynthesis in vitro in the honeybee (Apis mellifera)

Caste-specific pheromone biosynthesis is a prerequisite for reproductive skew in the honeybee. Nonetheless, this process is not hardwired but plastic, in that egg-laying workers produce a queen-like pheromone. Studies with Dufour's gland pheromone revealed that, in vivo, workers' gland biosynthesis matches the social status of the worker, i.e., sterile workers showed a worker-like pattern whereas fertile workers showed a queen-like pattern (production of the queen-specific esters). However, when incubated in vitro, the gland spontaneously exhibits the queen-like pattern, irrespective of its original worker type, prompting the notion that ester production in workers is under inhibitory control that is queen-dependent. We tested this hypothesis by exposing queen or worker Dufour's glands in vitro to brain extracts of queens, queenright (sterile) workers and males. Unexpectedly, worker brain extracts activated the queen-like esters biosynthesis in workers' Dufour's gland. This stimulation was gender-specific; queen or worker brains demonstrated a stimulatory activity, but male brains did not. Queen gland could not be further stimulated. Bioassays with heated and filtered extracts indicate that the stimulatory brain factor is below 3,000 Da. We suggest that pheromone production in Dufour's gland is under dual, negative-positive control. Under queenright conditions, the inhibitor is released and blocks ester biosynthesis, whereas under queenless conditions, the activator is released, activating ester biosynthesis in the gland. This is consistent with the hypothesis that queenright workers are unequivocally recognized as non-fertile, whereas queenless workers try to become "false queens" as part of the reproductive competition.

The mechanism of the queen signal in regulation of worker reproduction in the myrmicine ant Aphaenogaster smythiesi japonica

In ants, it is generally accepted that worker reproduction is regulated by a chemical signal informing workers of the queen's presence and stimulating workers to refrain from their own reproduction. This is known as the ‘queen signal’ — the mechanism for which is not well understood. In the myrmicine ant, Aphaenogaster smythiesi japonica, the queen signal mechanism was investigated by laboratory experiments. Firstly, in artificial nests where the nest chambers were separated by meshes, the workers were kept under queenright and queenless conditions in the laboratory. After 1 month in queenless conditions, a remarkable development of ovarioles with reproductive eggs was observed in a few queenless workers of each colony. Such development was not observed in queenright workers. This indicates that information of queen presence cannot be transmitted through meshes and that the chemical substances transmitting queen signal are not volatile. Secondly, in other artificial nests, the contact with the queen by workers was limited by attaching a piece of plastic string to their dorsal alitrunk and connecting nest chambers with small tubes. According to the limitation of contact, the workers were classified into three groups, queenright workers (Q+), queenless workers (Q–) and workers which could freely contact with the queen and all workers (FM). The development of ovaries with reproductive eggs was never observed in all workers of the three groups Q+, Q– and FM, indicating that their reproduction was regulated. This strongly suggests that A. s. japonica workers could recognize the queen's presence by direct contact with the queen and that the information could be transmitted via workers in the colony.

The initial stages of oogenesis and their relation to differential fertility in the honey bee ( Apis mellifera) castes

Neither the overall differences in ovariole number nor the caste-specifically modulated expression of vitellogenin can fully explain the striking caste differences in honey bee reproduction, in particular the mechanisms that block oogenesis in virgin queens and in workers kept in the presence of a queen. For this reason we investigated the initial stages of oogenesis in queens in relation to mating status and in workers exposed to different social conditions. A striking feature in ovarioles of both castes was a considerably elongated terminal filament which consisted not only of normal terminal filament cells but also contained apparently undifferentiated cells that were tentatively considered as stem cells. BrdU incorporation was detected in the upper germarium, as well as in the terminal filament. Cytoskeleton analysis by TRITC-phalloidin labeling for F-actin, and immunofluorescence detection for β-tubulin did not reveal structural differences in the early oogenesis steps between queens and queenless workers. In contrast, queenright workers showed signs of a disorganized microtubule and microfilament system that could explain the histological evidence for progressive cell death observed in the germaria. In addition to cytoplasmic tubulin we also detected marked intranuclear foci indicating the presence of nuclear β II-tubulin.

Food influence on colonial recognition and chemical signature between nestmates in the fungus-growing ant Acromyrmex subterraneus subterraneus

Neotropical “Fungus-growing” leaf-cutting ants (tribe Attini) live in obligatory symbiosis with a fungus, which they grow on fresh leaves harvested by workers. Colonial recognition is likely based on chemical cues provided by cuticular hydrocarbons that have been found to be partly influenced by environmental odor sources. The diet breadth of Acromyrmex subterraneus subterraneus enabled us to test the impact of different plant diets on colonial recognition. The intermediary of the fungus in the ants’ feeding habit adds a special angle to the question. From a queenright (QR) mother colony of A. s. subterraneus we formed several groups of queenless (QL) workers with fungus (approx. 700 ants). The QR colony and two of the QL-groups were fed with the same diet of fresh bramble leaves. Two other QL-groups were fed with privet leaves and two with rose flowers. After 4 months, QR workers were significantly more aggressive towards the QL-group fed with rose flowers or privet leaves than towards workers of the QL-groups fed with fresh bramble leaves. Rose-fed QL workers were aggressive towards privet-fed QL workers and vice versa, but never towards workers of their counterpart group that fed on the same diet. These results suggest that the absence of the queen or the separation time between groups played a minor role in shaping nestmate recognition cues as compared to the diet. The behavioral studies were supplemented by chemical analyses of cuticles, postpharyngeal glands (PPG) and plant-food extracts revealing profiles variations that were correlated with the dietary changes. However, although the plant extract contained several hydrocarbons there was no congruency between the plant profile and the respective diet-group ants. These results support the hypothesis that the diet influences indirectly the chemical profiles and consequently the recognition cues in A. s. subterraneus.

Odour convergence and tolerance between nestmates through trophallaxis and grooming in the ant Camponotus fellah (Dalla Torre)

Summary. Social isolation provides a useful tool to study nestmate recognition in ants. In Camponotus fellah, reintroduction of 10-day isolated (IS) workers to their colony resulted in intensive trophallaxis and grooming, while longer isolation periods generally provoked rejection of the IS ants. In the first experiment the behaviour of queenless (QL) and queenright (QR) workers towards 10-day IS workers was tested. Trophallaxis of QL or QR with IS workers was of similar magnitude, but was significantly higher than that among the QL or QR, or that between QL and QR workers. Allogrooming was mostly initiated by the resident non-isolated ants (QL or QR) possibly because they detected a slight mismatch between the IS ant’s odour and their own template, which represents the group odour. It appears that the presence/absence of the queen did not affect nestmate recognition cues of workers. The second experiment demonstrated that 20-day IS workers were strongly aggressed by colony guards, irrespective of whether they were QL or QR. However, if they were permitted to exchange trophallaxis and grooming with 5 young nestmates (companion ants) for 5 days before reintroduction to their colony, aggression was greatly reduced, irrespective of the origin of the companion ants (QR or QL). Chemical analysis showed a significant divergence between the hydrocarbon profiles of IS and both QL and QR groups, but a prior contact of the IS workers with companion ants resulted in re-convergence of their profile with that of the colony. These results demonstrate that nestmate recognition cues are exchanged between workers via trophallaxis and grooming and that they are not dominated by queen cues, two conditions that fulfil Gestalt nestmate recognition signals requirements.

Different policing rates of eggs laid by queenright and queenless anarchistic honey-bee workers ( Apis mellifera L.)

Worker-reproduction is rare in queenright honey-bee colonies. When workers do lay eggs, their eggs are normally eaten by other workers presumably because they lack the queen's egg-marking signal. Workers use the absence of this queen signal to enforce the queen's reproductive monopoly by policing any worker-laid eggs. In contrast, in anarchistic colonies, the majority of the males arise from worker-laid eggs. Anarchistic worker-laid eggs escape policing because workers perceive anarchistic eggs as queen-laid. However, in this study, we show that eggs laid by queenless anarchistic workers do not escape policing and have very similar removal rates to worker-laid eggs from queenless wild-type (i.e. non-anarchistic) colonies. This suggests that, under queenless conditions, eggs laid by anarchistic workers lose their chemical protection and are therefore no longer perceived as queen-laid. Hence, the egg-marking signal seems to be only applied to eggs when queen and brood are present. This suggests that in the absence of queen and brood, the biosynthetic pathway that produces the egg-marking signal is switched off.

Colony insularity through queen control on worker social motivation in ants.

We investigated the relative contribution of the queen and workers to colony nestmate recognition cues and on colony insularity in the Carpenter ant Camponotus fellah. Workers were either individually isolated, preventing contact with both queen and workers (colonial deprived, CD), kept in queenless groups, allowing only worker-worker interactions (queen deprived, QD) or in queenright (QR) groups. Two weeks post-separation QD and QR workers were amicable towards each other but both rejected their CD nestmates, which suggests that the queen does not measurably influence the colony recognition cues. By contrast, aggression between QD and QR workers from the same original colony was apparent only after six months of separation. This clearly demonstrates the power of the Gestalt and indicates that the queen is not a dominant contributor to the nestmate recognition cues in this species. Aggression between nestmates was correlated with a greater hydrocarbon (HC) profile divergence for CD than for QD and QR workers, supporting the importance of worker-worker interactions in maintaining the colony Gestalt odour. While the queen does not significantly influence nestmate recognition cues, she does influence colony insularity since within 3 days QD (queenless for six months) workers from different colony origins merged to form a single queenless colony. By contrast, the corresponding QR colonies maintained their territoriality and did not merge. The originally divergent cuticular and postpharyngeal gland HC profiles became congruent following the merger. Therefore, while workers supply and blend the recognition signal, the queen affects worker-worker interaction by reducing social motivation and tolerance of alien conspecifics.

Honeybees Dufour's gland - idiosyncrasy of a new queen signal

Dufour's gland (DG) secretion of queens in Apis mellifera is normally caste specific. In queenright (QR) workers it is composed of odd n alkanes, while in queens it also possesses long chain esters. However, glandular expression is plastic since queenless (QL) workers produce a queen-like secretion. More- over, QR gland incubated in vitro produced these esters, indicating that glandular activity is regulated. We tested the hypothesis that the secretion is an egg marking pheromone. Chemical analysis of the egg coating re- vealed minute amounts of the queen esters, but neither queen secretion nor the synthetic esters were able to protect worker-laid eggs from policing, refuting the hypothesis. Analysis of abdominal tips further revealed that Dufour's egg secretion is also smeared on the abodominal cuticle, suggesting that its presence on egg sur- face may be due to passive contamination. Next, we tested the hypothesis that the secretion serves as a queen signal. Indeed queens, but not worker glandular secretion were attractive to workers. Dufour's gland / honeybee, queen-signal / caste-specificity / retinue behavior

Plasticity in caste-related exocrine secretion biosynthesis in the honey bee ( Apis mellifera)

Plasticity of Dufour's gland secretion in the honey bee is correlated with the individual's plasticity. Queens and queenless (QL) egg-laying workers possess a bouquet of esters and hydrocarbons, whereas queenright (QR) workers produce exclusively hydrocarbons. The effects of social environment (QR vs. QL conditions) and possible physiological constraints on the gland were studied by following the biosynthesis of these classes of compounds in vivo and in vitro. Biosynthesis in vivo followed the prediction based on glandular chemistry. Queens and QL egg-laying workers, but not QR workers or QL foragers, showed incorporation of sodium acetate into both hydrocarbons and esters. In contrast, the in vitro studies revealed that, in addition to queens and QL egg-laying workers, QR nurses retained their ability to produce the queen characteristic esters. Although there was some ester production in foragers, it occurred to a lesser extent. It is possible that the glands in the older foragers undergo irreversible changes. The in vitro incubation also revealed a temporal activation of ester biosynthesis in QR workers. In these glands alcohols, corresponding to the alcohol moiety of the esters, predominated in short-term incubations but decreased as the amount of newly synthesized esters increased. In contrast, queens and QL egg-laying workers showed predominant incorporation into esters from the onset of incubation. Thus, expression within the workers' Dufour's gland is regulated. In the presence of a queen, ester production is inhibited. Once the queen is removed the physiologically unconstrained gland starts to biosynthesize the queen-specific esters after a certain lag needed for the build-up of precursors and the enzymatic machinery.

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.

Juvenile hormone titers, juvenile hormone biosynthesis, ovarian development and social environment in Bombus terrestris

The effects of the social environment and age on juvenile hormone (JH) and reproduction were investigated by measuring ovarian development, hemolymph levels of JH III, and rates of JH biosynthesis from the same individual bumble bees ( Bombus terrestris). Differences in social environment were associated with differences in rates of JH biosynthesis, JH titer and ovarian development. Young queenless workers had a higher rate of JH biosynthesis, JH titer and ovarian development than queenright (QR) workers of similar age. Dominant workers in QR colonies had a higher rate of JH biosynthesis, JH titer and ovarian development than low ranked workers of similar size. There was a positive correlation between JH titer and ovarian development, but no correlation between rate of JH biosynthesis and ovarian development or between JH biosynthesis and JH titer. Both JH titer and rate of JH biosynthesis increased with age from emergence to 3 days of age, but 6-day-old workers, egg-laying workers, and actively reproducing queens had high JH titers and highly developed ovaries but low rates of JH biosynthesis. These results show that reproduction in B. terrestris is strongly affected by the social environment and the influence of the environment on reproduction is mediated by JH. Our data also indicate that the rate of JH biosynthesis measured in vitro is not a reliable indicator of JH titer or ovarian development in B. terrestris; possible reasons are discussed.