MicroRNA ame-let-7 targets Amdop2 to increase sucrose sensitivity in honey bees (Apis mellifera).

BACKGROUND: As an important catecholamine neurotransmitter in invertebrates and vertebrates, dopamine plays multiple roles in the life of the honey bee. Dopamine receptors (DA), which specifically bind to dopamine to activate downstream cascades, have been reported to be involved in honey bee reproduction, division of labour, as well as learning and motor behaviour. However, how dopamine receptors regulate honey bee behavior remains uninvestigated. RESULTS: The expression level of Amdop2 in the brain increased with the age of worker bees, which was just the opposite trend of ame-let-7. Inhibition of ame-let-7 through feeding an inhibitor upregulated Amdop2 expression; conversely, overexpression of ame-let-7 through a mimic downregulated Amdop2. Moreover, knockdown of Amdop2 in forager brain led to significantly higher sucrose responsiveness, which is similar to the phenotype of overexpression of ame-let-7. Finally, we confirmed that ame-let-7 directly targets Amdop2 in vitro by a luciferase reporter assay. CONCLUSIONS: ame-let-7 is involved in the dopamine receptor signaling pathway to modulate the sucrose sensitivity in honey bees. Specifically, it down-regulates Amdop2, which then induces higher responses to sucrose. These results further unraveled the diverse mechanisms of the dopamine pathway in the regulation of insect behavior.

lncRNA profile of Apis mellifera and its possible role in behavioural transition from nurses to foragers.

BACKGROUND: The behavioural transition from nurses to foragers in honey bees is known to be affected by intrinsic and extrinsic factors, including colony demography, hormone levels, brain chemistry and structure, and gene expression in the brain. However, the molecular mechanism underlying this behavioural transition of honey bees is still obscure. RESULTS: Through RNA sequencing, we performed a comprehensive analysis of lncRNAs and mRNAs in honey bee nurses and foragers. Nurses and foragers from both typical colonies and single-cohort colonies were used to prepare six libraries to generate 49 to 100 million clear reads per sample. We obtained 6863 novel lncRNAs, 1480 differentially expressed lncRNAs between nurses and foragers, and 9308 mRNAs. Consistent with previous studies, lncRNAs showed features distinct from mRNAs, such as shorter lengths, lower exon numbers, and lower expression levels compared to mRNAs. Bioinformatic analysis showed that differentially expressed genes were mostly involved in the regulation of sensory-related events, such as olfactory receptor activity and odorant binding, and enriched Wnt and FoxO signaling pathways. Moreover, we found that lncRNAs TCONS_00356023, TCONS_00357367, TCONS_00159909 and mRNAs dop1, Kr-h1 and HR38 may play important roles in behavioural transition in honey bees. CONCLUSION: This study characterized the expression profile of lncRNAs in nurses and foragers and provided a framework for further study of the role of lncRNAs in honey bee behavioural transition.

Socially selected ornaments influence hormone titers of signalers and receivers.

Decades of behavioral endocrinology research have shown that hormones and behavior have a bidirectional relationship; hormones both influence and respond to social behavior. In contrast, hormones are often thought to have a unidirectional relationship with ornaments. Hormones influence ornament development, but little empirical work has tested how ornaments influence hormones throughout life. Here, we experimentally alter a visual signal of fighting ability in Polistes dominulus paper wasps and measure the behavioral and hormonal consequences of signal alteration in signalers and receivers. We find wasps that signal inaccurately high fighting ability receive more aggression than controls and receiving aggression reduces juvenile hormone (JH) titers. As a result, immediately after contests, inaccurate signalers have lower JH titers than controls. Ornaments also directly influence rival JH titers. Three hours after contests, wasps who interacted with rivals signaling high fighting ability have higher JH titers than wasps who interacted with rivals signaling low fighting ability. Therefore, ornaments influence hormone titers of both signalers and receivers. We demonstrate that relationships between hormones and ornaments are flexible and bidirectional rather than static and unidirectional. Dynamic relationships among ornaments, behavior, and physiology may be an important, but overlooked factor in the evolution of honest communication.

Rapid juvenile hormone downregulation in subordinate wasp queens facilitates stable cooperation.

In many cooperatively breeding animals, subordinate group members have lower reproductive capacity than dominant group members. Theory suggests subordinates may downregulate their reproductive capacity because dominants punish subordinates who maintain high fertility. However, there is little direct experimental evidence that dominants cause physiological suppression in subordinates. Here, we experimentally test how social interactions influence subordinate reproductive hormones in Polistes dominula paper wasps. Polistes dominula queens commonly found nests in cooperative groups where the dominant queen is more fertile than the subordinate queen. In this study, we randomly assigned wasps to cooperative groups, assessed dominance behaviour during group formation, then measured levels of juvenile hormone (JH), a hormone that mediates Polistes fertility. Within three hours, lowest ranking subordinates had less JH than dominants or solitary controls, indicating that group formation caused rapid JH reduction in low-ranking subordinates. In a second experiment, we measured the behavioural consequences of experimentally increasing subordinate JH. Subordinates with high JH-titres received significantly more aggression than control subordinates or subordinates from groups where the dominant's JH was increased. These results suggest that dominants aggressively punished subordinates who attempted to maintain high fertility. Low-ranked subordinates may rapidly downregulate reproductive capacity to reduce costly social interactions with dominants. Rapid modulation of subordinate reproductive physiology may be an important adaptation to facilitate the formation of stable, cooperative groups.

Preservation of orange juice using propolis.

Orange juice is one of the most popular and the most consumed fruit juices all over the world, especially in Europe and the chemical food preservatives, such as sodium benzoate, potassium sorbate and their mixtures, have long been used in orange juice sold on the market. Excessive consumption of these preservatives may be hazardous to human health. Propolis, composed of resins collected from plant buds and exudates and mixed with salivary gland secretions and beeswax by honey bee workers, has been used as a human medicine and natural food preservative. We hypothesis that propolis, without alcohol, can serve as an alternative and non-synthetic preservative of orange juice. In this study, the preservative effect of propolis emulsion on orange juice was determined up to 35 days. Propolis emulsion (0.02 g/mL propolis, 12 mL), emulsion control (12 mL containing Tween-80, hydrophilic phospholipid and polyethylene glycol 400), sodium benzoate (0.4 g) and potassium sorbate (0.4 g) was each added to 388, 388, 400 and 400 mL orange juice respectively. Propolis emulsion showed significant inhibition of bacteria growth and l-ascorbic acid degradation. Orange juice pH value, titratable acidity, total phenolic content, color and antioxidant capacity were effectively maintained by propolis emulsion. A control solution with all the same emulsifying agents without propolis did not show these properties. It was concluded that propolis can be used as a natural additive agent in orange juice or other fruit juices as an alternative to chemical preservatives.

No effect of juvenile hormone on task performance in a bumblebee (Bombus terrestris) supports an evolutionary link between endocrine signaling and social complexity.

A hallmark of insect societies is a division of labor among workers specializing in different tasks. In bumblebees the division of labor is related to body size; relatively small workers are more likely to stay inside the nest and tend ("nurse") brood, whereas their larger sisters are more likely to forage. Despite their ecological and economic importance, very little is known about the endocrine regulation of division of labor in bumblebees. We studied the influence of juvenile hormone (JH) on task performance in the bumblebee Bombus terrestris. We first used a radioimmunoassay to measure circulating JH titers in workers specializing in nursing and foraging activities. Next, we developed new protocols for manipulating JH titers by combining a size-adjusted topical treatment with the allatotoxin Precocene-I and replacement therapy with JH-III. Finally, we used this protocol to test the influence of JH on task performance. JH levels were either similar for nurses and foragers (three colonies), or higher in nurses (two colonies). Nurses had better developed ovaries and JH levels were typically positively correlated with ovarian state. Manipulation of JH titers influenced ovarian development and wax secretion, consistent with earlier allatectomy studies. These manipulations however, did not affect nursing or foraging activity, or the likelihood to specialize in nursing or foraging activity. These findings contrast with honeybees in which JH influences age-related division of labor but not adult female fertility. Thus, the evolution of complex societies in bees was associated with modifications in the way JH influences social behavior.

Genetic architecture of a hormonal response to gene knockdown in honey bees.

Variation in endocrine signaling is proposed to underlie the evolution and regulation of social life histories, but the genetic architecture of endocrine signaling is still poorly understood. An excellent example of a hormonally influenced set of social traits is found in the honey bee (Apis mellifera): a dynamic and mutually suppressive relationship between juvenile hormone (JH) and the yolk precursor protein vitellogenin (Vg) regulates behavioral maturation and foraging of workers. Several other traits cosegregate with these behavioral phenotypes, comprising the pollen hoarding syndrome (PHS) one of the best-described animal behavioral syndromes. Genotype differences in responsiveness of JH to Vg are a potential mechanistic basis for the PHS. Here, we reduced Vg expression via RNA interference in progeny from a backcross between 2 selected lines of honey bees that differ in JH responsiveness to Vg reduction and measured JH response and ovary size, which represents another key aspect of the PHS. Genetic mapping based on restriction site-associated DNA tag sequencing identified suggestive quantitative trait loci (QTL) for ovary size and JH responsiveness. We confirmed genetic effects on both traits near many QTL that had been identified previously for their effect on various PHS traits. Thus, our results support a role for endocrine control of complex traits at a genetic level. Furthermore, this first example of a genetic map of a hormonal response to gene knockdown in a social insect helps to refine the genetic understanding of complex behaviors and the physiology that may underlie behavioral control in general.

Comparative virulence and competition between Nosema apis and Nosema ceranae in honey bees (Apis mellifera).

Honey bees (Apis mellifera) are infected by two species of microsporidia: Nosema apis and Nosemaceranae. Epidemiological evidence indicates that N. ceranae may be replacing N. apis globally in A. mellifera populations, suggesting a potential competitive advantage of N. ceranae. Mixed infections of the two species occur, and little is known about the interactions among the host and the two pathogens that have allowed N. ceranae to become dominant in most geographical areas. We demonstrated that mixed Nosema species infections negatively affected honey bee survival (median survival=15-17days) more than single species infections (median survival=21days and 20days for N. apis and N. ceranae, respectively), with median survival of control bees of 27days. We found similar rates of infection (percentage of bees with active infections after inoculation) for both species in mixed infections, with N. apis having a slightly higher rate (91% compared to 86% for N. ceranae). We observed slightly higher spore counts in bees infected with N. ceranae than in bees infected with N. apis in single microsporidia infections, especially at the midpoint of infection (day 10). Bees with mixed infections of both species had higher spore counts than bees with single infections, but spore counts in mixed infections were highly variable. We did not see a competitive advantage for N. ceranae in mixed infections; N. apis spore counts were either higher or counts were similar for both species and more N. apis spores were produced in 62% of bees inoculated with equal dosages of the two microsporidian species. N. ceranae does not, therefore, appear to have a strong within-host advantage for either infectivity or spore growth, suggesting that direct competition in these worker bee mid-guts is not responsible for its apparent replacement of N. apis.

Ubx promotes corbicular development in Apis mellifera.

The key morphological feature that distinguishes corbiculate bees from other members of the Apidae family is the presence of the corbicula (pollen basket) on the tibial segment of hind legs. Here, we show that in the honeybee (Apis mellifera), the depletion of the gene Ultrabithorax (Ubx) by RNAi transforms the corbicula from a smooth, bristle-free concave structure to one covered with bristles. This is accompanied by a reduction of the pollen press, which is located on the basitarsus and used for packing the pollen pellet as well as a loss of the orderly arrangement of the rows of bristles that form the pollen comb. All these changes make the overall identity of workers' T3 legs assume that of the queen. Furthermore, in a corbiculate bee of a different genus, Bombus impatiens, Ubx expression is also localized in T3 tibia and basitarsus. These observations suggest that the evolution of the pollen gathering apparatus in corbiculate bees may have a shared origin and could be traced to the acquisition of novel functions by Ubx, which in Apis were instrumental for subsequent castes and behavioural differentiation.

Large cells suppress the reproduction of Varroa destructor.

BACKGROUND: The parasitic mite, Varroa destructor has posed a threat to the health and survival of European honey bees, Apis mellifera worldwide. There is a prevailing belief that small comb cells could provide a management tool against Varroa mites. However, the hypothesis that smaller cells can impede Varroa reproduction has not been fully tested. Here, we tested this hypothesis under laboratory conditions by using two distinct Varroa in vitro rearing systems: one involved gelatin capsules of different sizes, specifically size 00 (0.95 mL) versus size 1 (0.48 mL), and the second consisted of brood comb cells drawn on 3D printed foundations with varying cell sizes, ranging from 5.0 mm to 7.0 mm at 0.5 mm intervals. RESULTS: The results showed that mother mites in size 00 cells had significantly lower fecundity and fertility compared to those in size 1 cells. Interestingly, the reproductive suppression in larger cells could be reversed by adding an extra worker larva. Similarly, gonopore size of mother mites was smaller in size 00 cells, but restored with another host larva. Furthermore, both the fecundity and fertility of mother mites decreased linearly with the size of brood comb cells. CONCLUSIONS: Our results suggest that the reproduction of V. destructor is hindered by larger cells, possibly because larger brood cells disperse or weaken host volatile chemical cues that are crucial for Varroa reproduction. The insights derived from this study are expected to hold significant implications for the implementation of Varroa management programs. © 2024 Society of Chemical Industry.

Genomewide analysis indicates that queen larvae have lower methylation levels in the honey bee (Apis mellifera).

The honey bee is a social insect characterized by caste differentiation, by which a young larva can develop into either a queen or a worker. Despite possessing the same genome, queen and workers display marked differences in reproductive capacity, physiology, and behavior. Recent studies have shown that DNA methylation plays important roles in caste differentiation. To further explore the roles of DNA methylation in this process, we analyzed DNA methylome profiles of both queen larvae (QL) and worker larvae (WL) of different ages (2, 4, and 6 day old), by using methylated DNA immunoprecipitation-sequencing (meDIP-seq) technique. The global DNA methylation levels varied between the larvae of two castes. DNA methylation increased from 2-day- to 4-day-old QL and then decreased in 6-day-old larvae. In WL, methylation levels increased with age. The methylcytosines in both larvae were enriched in introns, followed by coding sequence (CDS) regions, CpG islands, 2 kbp downstream and upstream of genes, and 5' and 3' untranslated regions (UTRs). The number of differentially methylated genes (DMGs) in 2-, 4-, and 6-day-old QL and WL was 725, 3,013, and 5,049, respectively. Compared to 4- and 6-day-old WL, a large number of genes in QL were downmethylated, which were involved in many processes including development, reproduction, and metabolic regulation. In addition, some DMGs were concerned with caste differentiation.

Nosema ceranae induced mortality in honey bees (Apis mellifera) depends on infection methods.

Nosema ceranae infection can reduce survival of the Western honey bee, Apis mellifera, but experiments examining its virulence have highly variable results. This variation may arise from differences in experimental techniques. We examined survival effects of two techniques: Nosema infection at day 1 without anesthesia and infection at day 5 using CO2 anesthesia. All bees infected with the latter method had poorer survival. Interestingly, these bees also had significantly fewer spores than bees infected without anesthesia. These results indicate that differences in Nosema ceranae-induced mortality in honey bees may be partially due to differences in experimental techniques.

EAG responses of Apis cerana to floral compounds of a biodiesel plant, Jatropha curcas (Euphorbiaceae).

The Eastern honey bee, [Apis cerana (F.)], is an important and common pollinator for an important biodiesel tree, [Jatropha curcas (L.)]. To understand sensitivity of A. cerana to different floral compounds, we quantified volatile floral compounds of J. curcas, then determined electroantennogram (EAG) responses of A. cerana to 11 compounds each at five doses (0.4, 4, 40, 400, and 4,000 microg) of six most active floral compounds. Our results demonstrated that floral compounds of J. curcas differ in variety and quantity while linalool is always a major constituent in floral blends from three different plantations. Antennae of A. cerana responded to all 11 floral compounds, implying a broad sensitivity of A. cerana to different floral compounds of J. curcas. Antennae of A. cerana were most sensitive to six compounds, including all aldehydes (decanal, hexanal, nonanal, and octanal), linalool, and an alcohol (3-hexenol), suggesting that A. cerana possesses chemoreceptors to aldehydes, linalool, and alcohol on the antenna. Furthermore, low doses elicited a zero EAG response and high doses a positive one under all of six most active compounds. Thus, EAG responses of A. cerana were both chemical specific and dose-dependent. Our results here suggest that A. cerana is senstive to various floral compounds, and linalool in the floral blends of J. curcas plays a key role to attract A. cerana.

Mblk-1 regulates sugar responsiveness in honey bee (Apis mellifera) foragers.

Brain transcriptional regulatory network for behavior demonstrates that brain gene expression in the honey bee can be accurately predicted from the expression transcription factors (TFs), but roles for specific TFs are less understood. Mushroom bodies (MBs) are important for learning, memory and sensory integration in the honey bee brain. A TFs, Mblk-1, expressed preferentially in the large-type Kenyon cells of the honeybee MBs is predicted to be involved in brain function by regulating transcription of its target genes in honey bee. However, its function and the mechanism of regulation in behavior of honey bee is still obscure. Here we show that Mblk-1 had significantly higher expression in the brains of forager bees relative to nurse bees. Mblk-1 was significantly inhibited in bees fed small interfering RNA. In addition, inhibition of Mblk-1 decreased sucrose responsiveness in foragers. Finally, we determined that Mblk-1 regulated the messenger RNA of AmGR1. These findings suggest that Mblk-1 may target AmGR1 to regulate the sucrose responsiveness of foragers.

The cell invasion preference of Varroa destructor between the original and new honey bee hosts.

Honey bees (Apis) are important pollinators for food crops and wild plants, but are facing great threats from pathogens and parasites, especially an obligate ectoparasitic mite, Varroa destructor. Cell invasion is a key step for V. destructor to reproduce, and the parasite displays remarkable host preference in this process. Varroa destructor made its host-shift from its original host, the Asian honey bee Apis cerana, to the new host, the European honey bee Apis mellifera several decades ago. However, it remains largely unstudied whether V. destructor shows a cell invasion preference between the two host species. Using cell invasion bioassays on a modified four-well arena, we showed that V. destructor significantly preferred to invade the worker and drone larvae of A. mellifera rather than A. cerana, suggesting that the new host is much more attractive to the parasite than the original one. Using gas chromatography-mass spectrometry (GC-MS), we revealed significant differences between the cuticular hydrocarbon (CHC) profiles of worker and drone larvae of the two bee hosts. The amounts of methyl-branched alkanes and alkenes (unsaturated CHCs), but not n-alkanes, were significantly different, and A. mellifera worker and drone larvae were found to express significantly higher amounts of methyl-alkanes, while A. cerana larvae produced higher amounts of alkenes. Cell invasion bioassays with glass dummies showed that the mites preferred the glass dummies coated with the CHCs of A. mellifera worker or drone larvae, which indicates a role of larval CHCs in mediating the preferential cell invasion of Varroa. The findings from this study extend our understanding of the host preference of V. destructor, and can potentially contribute to the development of effective strategies for mite control.

The Role of Honey Bee Derived Aliphatic Esters in the Host-Finding Behavior of Varroa destructor.

Varroa destructor is an obligate ectoparasite of honey bees and shifted from its original host Apis cerana to the new host Apis mellifera in the first half of the twentieth century. The host shift has resulted in a great threat to the health and survival of A. mellifera colonies worldwide. Chemical signals play a crucial role in all aspects of the Varroa life cycle, including host finding. However, the chemical cues that affect the host finding behavior of Varroa mites are still not fully understood. In this study, we systematically profiled the headspace volatiles of both worker and drone larvae of the two honey bee species by using solid phase micro-extraction coupled to gas chromatography-mass spectrometry (SPME-GC-MS), and then used electrophysiological recording and Y-tube olfactometer bioassay to study the potential roles of the selected compounds. The chemical profiling showed that there were four aliphatic esters, ethyl myristate (EM), methyl palmitate (MP), ethyl palmitate (EP), and ethyl oleate (EO) commonly detected from all four types of larval hosts. Among them, EM was a new substance identified from honey bee headspace volatiles. Results from electrophysiological recordings indicated that all the aliphatic esters could elicit significant responses of Varroa pit organs on its forelegs. Moreover, behavioral analyses revealed that EM could significantly attract V. destructor at a medium dosage (10 µg), while MP had no observable effect on the mites and both EP and EO were able to repel the parasites. Our findings suggest an important role of host-derived aliphatic esters in Varroa host finding, and provide new chemicals for Varroa monitoring and control.

Honey Bee (Apis mellifera) Exposure to Pesticide Residues in Nectar and Pollen in Urban and Suburban Environments from Four Regions of the United States.

The risk of honey bee (Apis mellifera L.) exposure to pesticide residues while foraging for nectar and pollen is commonly explored in the context of agroecosystems. However, pesticides are also used in urban and suburban areas for vegetation management, vector control, and the management of ornamental plants in public and private landscapes. The extent to which pesticides pose a health risk to honey bees in these settings remains unclear. We addressed this at a landscape scale by conducting pesticide residue screening analyses on 768 nectar and 862 pollen samples collected monthly over 2 years from honey bee colonies located in urban and suburban areas in eight medium to large cities in California, Florida, Michigan, and Texas (USA). A risk assessment was performed using the US Environmental Protection Agency's BeeREX model whenever an oral toxicity value was available for a compound. Chemical analyses detected 17 pesticides in nectar and 60 in pollen samples during the survey. Approximately 73% of all samples contained no detectable pesticide residues. Although the number of detections varied among the sampled regions, fewer pesticides were detected in nectar than in pollen. Per BeeREX, four insecticides showed a potential acute risk to honey bees: imidacloprid, chlorpyrifos, and esfenvalerate in nectar, and deltamethrin in nectar and pollen. In general, exposure of honey bees to pesticides via nectar and pollen collection was low in urban and suburban areas across the United States, and no seasonal or spatial trends were evident. Our data suggest that honey bees are exposed to fewer pesticides in developed areas than in agricultural ones. Environ Toxicol Chem 2022;41:991-1003. © 2022 SETAC.

Contribution of diurnal and nocturnal insects to the pollination of Jatropha curcas (Euphorbiaceae) in southwestern China.

Jatropha curcas L. (Euphorbiaceae) is being increasingly planted worldwide, but questions remain regarding its pollination biology. This study examined the contribution of diurnal and nocturnal insects to the pollination of monoecious J. curcas, through its floral biology, pollination ecology, and foraging behavior of potential pollinators. Nectar production of both male and female flowers peaked in the morning, declined in the afternoon, and rapidly bottomed during the night in all of their anthesis days. The diurnal visitors to the flowers of J. curcas are bees and flies, and the nocturnal visitors are moths. Flowers received significantly more visits by diurnal insects than by nocturnal insects. Through bagging flowers during night or day or both or exclusion, we compared fruit and seed production caused by diurnal and nocturnal pollinators. Both nocturnal and diurnal visitors were successful pollinators. However, flowers exposed only to nocturnal visitors produced less fruits than those exposed only to diurnal visitors. Thus, diurnal pollinators contribute more to seed production by J. curcas at the study site.

Functional expression of an arachnid sodium channel reveals residues responsible for tetrodotoxin resistance in invertebrate sodium channels.

Tetrodotoxin (TTX) is a potent blocker of voltage-gated sodium channels, but not all sodium channels are equally sensitive to inhibition by TTX. The molecular basis of differential TTX sensitivity of mammalian sodium channels has been largely elucidated. In contrast, our knowledge about the sensitivity of invertebrate sodium channels to TTX remains poor, in part because of limited success in functional expression of these channels. In this study, we report the functional characterization in Xenopus oocytes of the first non-insect, invertebrate voltage-gated sodium channel from the varroa mite (Varroa destructor), an ecto-parasite of the honeybee. This arachnid sodium channel activates and inactivates rapidly with half-maximal activation at -18 mV and half-maximal fast inactivation at -29 mV. Interestingly, this arachnid channel showed surprising TTX resistance. TTX blocked this channel with an IC(50) of 1 microM. Subsequent site-directed mutagenesis revealed two residues, Thr-1674 and Ser-1967, in the pore-forming region of domains III and IV, respectively, which were responsible for the observed resistance to inhibition by TTX. Furthermore, sequence comparison and additional amino acid substitutions suggested that sequence polymorphisms at these two positions could be a widespread mechanism for modulating TTX sensitivity of sodium channels in diverse invertebrates.

The challenge hypothesis in an insect: juvenile hormone increases during reproductive conflict following queen loss in Polistes wasps.

The challenge hypothesis was proposed as a mechanism for vertebrates to optimize their testosterone titers by upregulating testosterone during periods of aggressive competition. Here we test key predictions of the challenge hypothesis in an independently evolved endocrine system: juvenile hormone (JH) in a social insect. We assess how social conflict influences JH titers in Polistes dominulus wasps. Aggressive conflict was produced by removing the queen from some colonies (experimental) and a low-ranked worker from other colonies (control). Queen removal produced competition among workers to fill the reproductive vacancy. Workers upregulated their JH titers in response to this social conflict; worker JH titers were higher in queenless than in queenright colonies. Furthermore, JH titers were associated with an individual's ability to dominate rivals; the worker that took over as the replacement queen had a substantially higher JH titer than did other workers. Finally, JH titers were positively associated with aggression in queenless colonies, but there was no relationship between JH and aggression in stable, queenright colonies. Overall, these results match key predictions of the challenge hypothesis and parallel much of the work on testosterone in vertebrates. Social modulation of hormone titers is not confined to a particular endocrine system but is likely to be an adaptive feature of endocrine systems across diverse taxa.