A social bee can learn a novel queen pheromone

A social bee can learn a novel queen pheromone | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A social bee can learn a novel queen pheromone View ORCID Profile Etya Amsalem , View ORCID Profile Abraham Hefetz doi: https://doi.org/10.1101/2025.05.30.657092 Etya Amsalem 1 Pennsylvania State University, Department of Entomology, University Park , Pennsylvania, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Etya Amsalem For correspondence: eau6{at}psu.edu Abraham Hefetz 2 School of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University , Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Abraham Hefetz Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Many pheromone responses are hardwired into the insect’s nervous system and are essential for critical behaviors such as mating, alarm signaling, and trail following. Workers of social species are also assumed to respond innately to queen pheromones, leading to changes in behavior and reproductive physiology. However, accumulating evidence highlights the potential roles of learning and experience in responses to pheromones regulating reproductive division of labor. To examine if the response to queen pheromone can be learned, we introduced bumblebee workers ( Bombus terrestris ) to a new queen pheromone by treating the queen daily with floral scents unfamiliar to the workers: either anisyl alcohol or methyl anthranilate. We allowed perfumed queens to establish colonies and examined worker attraction and egg laying following daily exposure to these odors without the queen, both with and without brood as context. Workers preferred the odor they grew up with and exposure to learned odors without the queen influenced worker egg laying only in the presence of brood, surprisingly resulting in increased egg laying. Our study demonstrates that workers can modify their behavior after learning an odor associated with their queen or nest. These learned odors function as context-dependent releaser pheromones, influencing worker attraction and egg-laying behavior. Introduction Insect life is largely regulated by pheromones, which are especially important for maintaining reproductive division of labor between fecund queens and sterile workers in social insects. Such pheromones are thought to regulate worker fertility and promote the formation of cohesive social groups where most females refrain from reproduction and act as helpers. Despite extensive research, only a handful of reproduction-regulating pheromones have been identified in social insects ( Amsalem, 2020 ; Hefetz, 2019 ; Liebig & Amsalem, 2025 ). This limitation is due to several factors, such as the overemphasis on the queen as the main driver of reproductive inhibition in the colony ( Liebig & Amsalem, 2025 ) while neglecting contextual elements ( Orlova & Amsalem, 2019 ), and the assumption that pheromonal responses are innate and hard-wired into the insect nervous system. This narrow framing can limit the scope of discovery and underrepresent the diversity of pheromonal systems, leading researchers to overlook the complexity and flexibility of pheromone communication. Indeed, many pheromonal responses are innate: male moths instinctively respond to pheromones released by conspecific females without prior exposure ( Martin & Hildebrand, 2010 ), ants follow pheromone trails instinctively to locate food or return to the nest ( Wilson, 1962 ), and honeybees respond to isopentyl acetate alarm pheromone component that elicits a stinging defense behavior in face of a threat ( Free, 1987 ). However, a growing body of evidence shows that responses to pheromones can be learned, modified or overridden by experience ( Beny & Kimchi, 2014 ). When reared in a foster nest, stingless bees of the species Scaptotrigona subobscuripennis follow the trail pheromones of their foster colony rather than those of their natal colony ( Reichle et al., 2013 ), male fruit flies innately reduce their attraction to females marked with cis-vaccenyl acetate but will further suppress their attraction through learning after being rejected by a mated female ( Ejima, Smith, Lucas, Levine, & Griffith, 2005 ; Ejima et al., 2007 ), honeybee workers modify their response to Nasonov gland pheromone (geraniol and citral) from appetitive to aversive when they learn to associate it with an electric shock ( Roussel, Padie, & Giurfa, 2012 ), and butterflies are able to learn a new sex pheromone ( Dion, Pui, Weber, & Monteiro, 2020 ). While many insect species are capable of learning ( Dukas, 2008 ; Leadbeater & Chittka, 2007 ), and pheromonal responses can be altered through experience, innate responses to pheromones involved in reproduction and social regulation may be especially susceptible to modification. Queen pheromones, for example, regulate worker reproduction by either manipulating or providing information that workers use to adjust their own reproductive behaviors ( Keller & Nonacs, 1993 ), and they differ from other pheromones in several ways that can increase flexibility in pheromonal response. First, they are competitive in nature ( West-Eberhard, 1984 ) and therefore more susceptible to cheating. Such pheromones often serve to resolve reproductive conflicts between the queen, who has a clear interest in being the sole reproducer, and the workers, who in some cases are capable of laying eggs that develop into males ( Bourke, 2001 ). Given the high fitness consequences for workers, additional forms of assurance may be required to prevent false signaling, for example, in cases where the queen falsely advertises her fecundity. Pheromones regulating reproduction are also highly context-dependent ( Orlova & Amsalem, 2019 ). For example, Bombus impatiens workers ignore the queen pheromones unless they are presented alongside young brood and/or visual cues from the queen ( Orlova & Amsalem, 2021 ). Context reinforces signal reliability and provides environmental relevance, and may also enable sensory integration, which may be essential for eliciting a response and enhancing overall effectiveness ( Orlova & Amsalem, 2019 ). Because context varies, responses to reproduction-mediating pheromones may be more easily shaped by learning and experience than other pheromonal systems. Indeed, it has been suggested that behaviors specific to time and place are more likely to depend on learning ( Dukas, 2008 ). Finally, reproductive pheromones also differ in signal design ( Endler, 1993 ); they are structurally diverse ( Amsalem, 2020 ; Liebig & Amsalem, 2025 ), generally less volatile and longer lasting in social insects, contributing to greater response flexibility and a stronger dependence on social experience and learning. Here, we examined whether the queen pheromone in Bombus terrestris can be learned. Bumblebees are an excellent species to examine the flexibility in pheromonal response since they are annual social bees exhibiting large flexibility in reproductive behavior ( Amsalem, Grozinger, Padilla, & Hefetz, 2015 ). Colonies are established by a single mated queen, develop through the spring and the summer, eventually producing sexuals and decline in the fall, during which only the mated queens survive and enter a winter diapause ( Amsalem et al., 2015 ). Following colony establishment, the young colony exhibits a complete reproductive division of labor between the fecund founder queen and the sterile workers, but approximately one month later workers start producing males from unfertilized eggs and engage in aggressive behavior towards each other and the queen ( Duchateau & Velthuis, 1988 ). Reproductive division of labor in bumblebee colonies is assumed to be regulated by various means, including context dependent queen pheromones ( Orlova & Amsalem, 2021 ; Orlova, Treanore, & Amsalem, 2020 ), the young brood ( Orlova, Starkey, & Amsalem, 2020 ; Santos, Galbraith, Starkey, & Amsalem, 2022 ; Starkey, Brown, & Amsalem, 2019 ; Starkey, Derstine, & Amsalem, 2019 ), signals produced by workers ( Amsalem, Kiefer, Schulz, & Hefetz, 2014 ; Amsalem, Twele, Francke, & Hefetz, 2009 ; Derstine et al., 2021 ; Orlova, Villar, Hefetz, Millar, & Amsalem, 2022 ) and other sociobiological factors including density ( Amsalem, Santos, Messner, & Murray, 2024 ) and the queen’s background ( Alaux, Jaisson, & Hefetz, 2005 ). We treated young founder queens daily by applying an external odor either anisyl alcohol (AA) or methyl anthranilate (MA) and allowed them to establish colonies. As colonies matured, workers were tested for behavioral preference between the odor they had developed with (hereafter, the ‘familiar odor’) or a novel odor (the alternative odor). We further tested for worker reproductive output in the absence of the queen following daily exposure to either the familiar or novel odors, either in the absence or presence of brood from their original colony (as a contextual signal). We hypothesized that if workers could learn to associate the externally applied odor with their queen’s pheromone, they would show attraction to the familiar odor as well as alter their reproductive physiology in its presence. We further hypothesized that the presence of a contextual signal (brood) would enhance the workers’ response to the externally applied odor and increase reproductive inhibition. Methods Bees Colonies of Bombus terrestris (n=8) were obtained from Poliam, Yad Mordechai, Israel on May 2024, a few days after the emergence of the first worker. All colonies were kept in incubators in darkness, 28° C and 50% RH, and bees were provided with a sucrose solution and fresh pollen purchased from Poliam, Yad Mordechai. At the experiment onset, all colonies contained a queen, 4-7 workers, and a small amount of brood at various stages that were housed in large wooden cages throughout the experiment (Supplementary material S1-A). From that point onward, queens were treated daily with 1 µl hexane containing 1 mg of either Anisyl alcohol (AA) or Methyl anthranilate (MA) (4 colonies per treatment). The solution was applied to the queen’s thorax using a pipette inserted into the colony with a minimum disturbance to the queens. Colony size was monitored daily, and sampling of workers (see below under “experimental design”) started once the colony reached 20 workers. Thereafter (35 days from its onset, in total), colony size was maintained at or near 20 workers. Treatment in all colonies started at least one week before the first workers were sampled and continued daily. Colonies were used as long as no signs of competition were observed. i.e., no worker reproduction or aggression, no multiple egg cells open at the same time and no presence of gyne larvae ( Amsalem et al., 2015 ). Experimental design The experiments examining the effect of applied odors on worker reproduction were conducted either without or in the presence of brood (experiments 1 and 2, respectively). In experiment 1 workers were sampled progressively between days 12 to 30, whereas in experiment 2, all workers were sampled on days 31-35. In both experiments, workers of random age were sampled in groups of 3, grouped according to their natal colonies, and placed in a small plastic cage for 7 days (Supplementary material S1-B). Callow workers, indicated by their silvery appearance, were not sampled. Thus, all workers had at least 2-3 days to interact with their queen prior to sampling. Colonies and cages were kept in separate incubators according to the odor treatment to prevent the exposure of volatiles of the alternate odor. Cages that were assigned a solvent control treatment were evenly split between the two incubators. All treatments were applied within the same time window, i.e., every morning between 9 to 11 am. In total, 171 cages of 3 workers were set in experiment 1 and 77 in experiment 2. Mortality of a single bee occurred in 14 cages (0.05% of the bees) and was randomly spread across treatments. All these cages were discarded, resulting in a total of 160 and 74 cages in experiments 1 and 2, respectively. The split of sample size per colony and treatment is provided in Supplementary material Table S1. Experiment 1 Cages of workers were randomly assigned to one of 3 treatments: 1) QL (queenless): these bees were provided with a clean dental wick daily with no treatment. These bees served as a positive control, showing the ability of workers to fully activate their ovaries within a week under QL conditions; 2) QL-familiar. These workers were provided daily with a dental wick treated with 1 µl hexane containing 1 mg of the same odor as in their natal colony. Workers from AA-treated queens were provided with AA whereas workers from MA-treated queens were provided with MA. These workers served to test whether they have learned the odor applied onto the queen and have recognized it as part of the queen pheromone and consequently are reproductively affected by it; 3) QL-novel. These workers were provided daily with a dental wick treated with a different odor than in their natal colonies (i.e., workers from AA-treated queens were provided with MA and vice versa). These workers served to test whether workers are reproductively affected by a novel odor which was not associated with their queen. All cages were kept for 7 days, after which they were scanned for the total number of eggs laid in them. In addition, we collected queenright (QR) bees from the mother colonies throughout the experiment and froze them immediately (11-15 bees per colony). These bees served as a negative control to assess the level of worker ovary activation in the natal queenright colonies. They were dissected, and their terminal oocytes’ size was measured. Experiment 2 Following the completion of the first experiment, the above mother colonies received a day or two without sampling to encourage their growth. On days 31-35 all workers from all colonies were placed in cages in groups of 3 nestmate workers together with a small amount of brood from their mother colony. The amount and developmental stage of the brood were recorded. Cages were randomly assigned to one of two treatments: 1) QL-familiar/Brood – these were provided daily with a dental wick treated with 1 µl hexane containing 1 mg of the same odor as in their natal colony; 2) QL-novel/brood. These workers were provided daily with a dental wick treated with a different odor than in their natal colonies. All cages were kept for 7 days, after which they were scanned for the total number of eggs laid in them. Behavioral assays During the first experiment, 15 workers from each colony were sampled for a behavioral test, examining their preference between the odor they grew up with versus the novel odor. Bioassays were conducted using a T-maze made of polycarbonate sheets (Supplementary video V1). The endpoint of each arm of the maze included a filter containing each of the two odors at the same concentration as applied to the queen. Each arm was connected into a plastic cage. Each worker was given 10 minutes to select the preferred arm, defined as positive if the worker passed the filter and entered the empty cage without the ability to return to the maze. Workers that did not decide within 10 minutes were defined as non-responsive. The odors were alternate between arms, and the maze was wiped clean prior to every bioassay. To prevent resampling of the same bee in consecutive bioassays, each tested worker was marked and returned to her colony at the end of the test. All tests were conducted within three days during the morning hours, about 20 days after the onset of the treatments of the queens, concurrently with the sampling of bees for experiment 1. Choice and chemical analysis of odors The odors in this study were chosen based on several parameters. Both odors were novel for the bees, yet abundant in their natural environment, which ensures in all probabilities that workers can perceive them. Anisyl alcohol (AA, CH3OC6H4CH2OH) and methyl anthranilate (MA, C8H9NO2) are floral scents and occur in plants that are pollinated by bees, including bumble bees, yet none of these compounds have been identified in bumble bees. In addition, the two compounds have different chemical structures (an alcohol and an ester, both containing aromatic rings) but that differ only slightly in their molecular weight (138.16 and 151.65 gram/mol in AA and MA, respectively), and volatility (boiling point 259 vs. 256, flesh point 110 vs. 104 and vapor pressure at 25° C is the same: 0.0+0.5 for both). To determine the daily concentration and frequency of application, we conducted an additional experiment where we applied 1 µl of hexane containing 1 mg of AA and MA to queenless workers in small groups and measured their quantity on the cuticle at timepoints 0, 24h, 48h and 72h post application. Workers (4-6 per timepoint, n=42) were frozen at −80° C. Their thorax was then immediately separated from the rest of their body and washed in hexane for 10 minutes. The thorax was gently removed from the solvent, and the extracts were analyzed using an Agilent 7890A GC equipped with a HP-5ms column (0.25id x 30m x 0.25 µm film thickness) connected to an Agilent 5975C mass spectrometer. The run was performed in splitless mode with temperature program from 60 °C at 10 °C/min up to 340 °C. The resulting chromatograms and spectra were analyzed using MSD ChemStation software (Agilent) and all peaks were identified using the NIST database and by comparing retention times and mass fragmentation with synthetic compound standards. Compounds were quantified using two internal standards: decanol and 2 undecanol, added to each extract at a concentration of 0.5 mg/1ul. As a control, we also quantified three cuticular hydrocarbons constituents (C23, C25 and C27) (Supplementary material Figure S2). Compound quantification was made in comparison to the two internal standards. Assessing worker reproduction Reproductive capacity of queenright workers was assessed by examining ovarian activation through dissection. Queenright workers from experiment 1 were frozen immediately after sampling and kept at −80°C. Each worker contains eight ovarioles (four in each ovary) and three of the largest terminal oocytes (at least one per ovary) were measured to the nearest micrometer. The sizes of the three largest oocytes were averaged and used as an index for ovarian activation. Reproduction by queenless workers was determined by counting the number of eggs laid. Since most cages that housed queenless workers contained eggs we assume that the workers had active ovaries and therefore skipped their dissection. All queenless cages were frozen after 7 days and eggs were counted per cage. Workers lay 2-4 eggs per cell and the cells are often merged (Supplementary material S1-C). Statistical analysis All analyses and visualizations were performed using JMP Pro 18. Quantification of cuticular compounds across time points was analyzed using a fixed-effects model followed by a Tukey post hoc test. To examine colony growth, we used a Linear Mixed Model (LMM) to compare the number of newly emerged workers over time during the growth and plateau phases, with colony ID included as a random effect. Ovarian activation in queenright workers across colonies and time was analyzed using a linear model with colony and colony age as fixed effects. To assess whether workers preferred the odor they were reared with, we performed Chi-square goodness-of-fit tests comparing the number of workers choosing each odor (AA or MA) against a null expectation of equal preference (50:50). Non-responding individuals were excluded from the analysis. To test the effect of treatment on the number of eggs laid, we used a Generalized Linear Mixed Model (GLMM) with a normal distribution. Colony ID, colony odor, colony age at the time of cage setup, and incubator identity (AA or MA) were included as random effects. To examine the combined effect of brood and odor on egg-laying counts, we used a GLMM with brood amount or developmental stage, treatment, and their interaction as fixed effects, and colony ID, colony odor, and colony age as random effects. Statistical significance was accepted at α = 0.05. Results Quantifying AA, MA and the three major cuticular hydrocarbon constituents on workers’ cuticle show that the two odors used in the study reached nearly zero amount within 24 hours from application (AA: F 3,17 =120.1, p<0.001; MA: F 3,17 =379.9, p<0.001; both followed by Tukey post-hoc test p<0.001 for timepoint 0 vs. all other timepoints) as opposed to the amounts of the three cuticular hydrocarbons that remain similar over time (C23: F 3,38 =1.24, p=0.3; C25: F 3,38 =2.11, p=0.11; C27: F 3,38 =1.46, p=0.23) ( Figure 2 ). Therefore, we chose to apply 1mg/1ul into the cuticle of queens daily. Download figure Open in new tab Figure 1. An overview of the experimental design: Queens from eight Bombus terrestris colonies were treated daily with either anisyl alcohol or methyl anthranilate shortly after the emergence of the first worker. Once colonies reached 20 workers, bees were sampled in groups of three and exposed to either the maternal colony’s odor (familiar) or a different odor (novel); queenless control groups received a solvent control. Additional queenright workers were sampled to assess colony reproductive status (not shown in the diagram). In a second experiment, groups of three workers were exposed to either a familiar or novel odor paired with brood from their maternal colony. Download figure Open in new tab Figure 2. Time-dependent GC/MS quantification of methyl anthranilate (MA), anisyl alcohol (AA), and three cuticular hydrocarbons (C23, C25, C27) from worker bees. Each compound was applied at a dose of 1 mg to the worker cuticle on day 0 and quantified using GC/MS at 0, 1, 2, and 3 days post-application. Quantities were normalized to two internal standards. All colonies developed normally, reaching 20 workers within 12–22 days ( Figure 3A ). There was a significant effect of colony age on worker number during the growth phase (F 1,108.4 =274.9, p < 0.001), indicating a consistent increase in population across colonies. No such effect was observed during the plateau phase (F 1,116.6 =1.82, p=0.17), when colonies were maintained at 20 workers. Ovarian activation of queenright workers did not vary over time (F 14,88 =1.21, p=0.28) or across colonies (F 7,88 =1.16, p=0.32) ( Figure 3B ). Download figure Open in new tab Figure 3. Colony growth and worker ovary activation over time in the eight colonies used in the study. Colonies reached 20 workers within 12–22 days from the start of the experiment (growth phase), which began a few days after the emergence of the first worker. In Experiment 1, bees were progressively sampled between days 12–30 and placed in cages for 7 days, while colony size was maintained below 20 workers throughout (the plateau phase). In Experiment 2, all bees and brood were collected on days 31– 35 (A). To assess the status of worker reproduction, workers were also sampled progressively from each colony between days 15–30, and their ovaries were measured immediately (B). Preference bioassays between the two odors showed that workers generally favored the odor they were reared with. Among colonies treated with MA, significant preferences for MA were observed in colony 2 (χ 2 = 3.77, p = 0.05) and colony 4 (χ 2 = 7.14, p = 0.007), while colonies 6 and 10 showed non-significant trends toward MA (χ 2 = 2.57, p = 0.11; χ 2 = 0.60, p = 0.44). Among colonies treated with AA, significant preferences for AA were found in colonies 5 (χ 2 = 5.40, p = 0.02), 7 (χ 2 = 4.57, p = 0.03), and 9 (χ 2 = 4.45, p = 0.03), but not in colony 11 (χ 2 = 0, p = 1.00) ( Figure 4 ). Download figure Open in new tab Figure 4. Two-choice olfactometer bioassays between methyl anthranilate (MA) and anisyl alcohol (AA) showing that workers preferred MA when reared with an MA-perfumed queen and AA when reared with an AA-perfumed queen (n = 15 workers per colony). Workers were given 10 minutes to make a choice before being classified as non-responsive. Non-response rates were low (0–13%) in all colonies except colony 11, where the rate was high (50%) and no clear preference was observed. The number of eggs laid within 7 days in the absence of brood did not differ between workers who were exposed to the same odor applied to their queen (familiar), or the novel odor, or were not exposed to any of the two odor treatments (GLMM, F 2,141.3 =0.15, p=0.85) ( Figure 5 ). The random factors tested (colony ID, colony odor, colony age at cage onset and incubator) had no impact on the model’s results. However, the number of eggs laid by workers varied across colonies (F 7,136 =6.52, p<0.001). Comparing the treatments within each colony did not result in significant effect of treatment (F 2,136 =0.3, p=0.73) or a significant interaction between colony and treatment (F 14,136 =0.91, p=0.54). Download figure Open in new tab Figure 5. Egg laying by workers exposed to methyl anthranilate, anisyl alcohol, or solvent control. Workers were housed in groups of three for 7 days, and their cages were treated daily with 1 mg of either the odor previously associated with their queen (familiar), a novel odor they had not encountered before, or hexane as a control. To examine the combined effect of odor treatment and brood on worker egg laying, we used three matrices for the brood: (1) the total number of brood at the end of the experiment; (2) the total number of young larvae at the end of the experiment; and (3) the brood developmental stage during the experiment as follows, EL means that the brood stage at onset was eggs that hatched as larvae during the 7 day experiment, LL are larvae who remained so throughout the experiment, and LP are larvae that pupated by the end of the experiment ( Starkey, Brown, et al., 2019 ). The number of worker-laid eggs within 7 days in the presence of brood was significantly affected by the treatment (GLMM, F 1,63.2 =4.91, p=0.03) and the amount of brood (F 1,68.8 =15.8, p=0.0002) but no interaction was found between the two (F 1,69.4 =0.06, p=0.79). Overall, brood amount negatively affected the number of eggs, but the workers exposed to a familiar odor lay slightly, but significantly more eggs compared to the workers exposed to the novel odor ( Figure 6A ). Running the model considering the number of young larvae at the end of the experiment resulted in significant differences as above for both treatments (F 1,63 =4.44, P=0.03) and the number of young larvae (F 1,68.4 =17.7, p<0.001), and no significant interactions (F 1,68.4 =0.14, p=0.7). Finally, running the model considering the developmental stage of the brood throughout the experiment resulted in a significant effect as above of brood developmental stage (F 1,65.6 =5.67, p=0.005), insignificant, yet close to significance effect of treatment (F 1,62.3 =3.15, p=0.08) and no significant interaction (F 1,65.4 =0.24, p=0.77). Young brood negatively affects the number of eggs ( Figure 6B ). Download figure Open in new tab Figure 6. Egg laying by workers exposed to methyl anthranilate or anisyl alcohol as a function of the total number of brood at the end of the experiment (A) and the developmental stage of the brood (B). Workers were kept in groups of three for 7 days with brood from their natal colonies. Cages were treated daily with 1 mg of either the odor previously associated with their queen (familiar) or a novel odor to which they had not been previously exposed. Discussion Pheromonal responses in insects are assumed to be innate and hard-wired, but a growing body of evidence shows that pheromones, especially these regulating reproductive behavior, can be learned or modified by experience ( Beny & Kimchi, 2014 ). In this study, we tested the hypothesis that perfuming the queen with an external odor, thus presumably creating a new pheromone composition, affects worker behavior and physiology and showed that bumble bee workers are indeed behaviorally influenced by such odors. Workers that grew up with a perfumed queen preferred the applied odor over a novel one in a T maze as well as altered their egg-laying behavior if exposed to the learned odor within context. However, it remains unclear whether the external odor was learned and encoded by workers as a general nest odor, after diffusing into the colony environment, or was embedded in worker’s brain as a component of the queen pheromone. If workers associate the odor with the queen’s presence and interpret it as a fertility or dominance signal, we expect the odor to suppress worker egg laying. However, the learned odor affected workers in the opposite direction, increasing the number of eggs they laid. A comparison with a previous study ( Orlova & Amsalem, 2021 ) may help interpret this finding. Orlova and Amsalem (2021) showed that worker reproduction in Bombus impatiens was reduced in the presence of queen pheromone, but only in a context-dependent manner, i.e., in combination with brood and a callow gyne. This supports the response we observed within context. However, the previous study tested only the combined effects of: (1) queen pheromonal secretion alone, which did not result in inhibited reproduction in workers; (2) queen pheromonal secretion and a callow gyne, which partially inhibited reproduction, and (3) queen pheromonal secretion, callow gyne, and brood, which fully suppressed worker reproduction. It did not test the effect of queen secretion and brood alone (like in the current study). It is plausible that, had this been tested, it would not have suppressed reproduction due to the absence of the visual cue provided by a callow gyne. Indeed, the absence of such a cue may signal to workers that the colony is entering the competition phase ( Duchateau & Velthuis, 1988 ), where the queen herself may be absent or compromised but queen odors and brood are still present, thus triggering worker reproduction before the end of the season. Visual stimuli were shown to be important to bumblebees, the absence of which led to increased volumes of the mushroom body lobes and calyces ( Jones, Leonard, Papaj, & Gronenberg, 2013 ). Another important difference between the two studies is the age of workers. In Orlova and Amsalem (2021) , workers were set in cages as callows, whereas in the current study, workers were of random age. In both studies, cages of workers were kept for 7 days. Neural development in bumble bees changes significantly as they age, and learning may be more efficient in younger bees ( Jones et al., 2013 ; Kraft, Spaethe, Rossler, & Groh, 2019 ). Honey bee workers, for instance, respond to the queen mandibular pheromone (QMP) only when young, while older workers tend to ignore it ( Vergoz et al., 2009 ; Vergoz, Schreurs, & Mercer, 2007 ). Thus, age may influence odor learning and the resulting behavioral outcomes. While workers clearly learned the novel odor, it remains unclear whether they encoded it as a queen-associated odor or a general nest-associated odor. This question warrants further investigation. Future studies could distinguish between these possibilities by applying the same odor to a dummy queen or to nest material alone and testing for similar behavioral responses, or by applying different odors to the queen and nest and assessing which workers learn and respond to. The effect of the learned odor likely falls within the definition of a releaser pheromone, which induces a behavioral but not a physiological change ( Wilson & Bossert, 1963 ). The odor altered worker attraction and egg-laying behavior but was unlikely to affect ovarian activation (ie, the size of oocyte size), as eggs were laid in most cages and thus most workers had fully activated ovaries regardless of treatment. However, treatments differed by the number of eggs laid in them. Although egg laying requires fully activated ovaries, it involves muscle contraction and is regulated separately from oogenesis and thus may also be considered a releaser effect. A clear releaser response to the learned odor is shown in Video 2, which captures an immediate, stereotypical excitation of workers following the introduction of the odor into the colony on a dental wick. Finally, it has been suggested that pheromones promoting reproductive behavior carry intrinsic reward value, which can be overridden by experience - particularly through associative learning. While many such modification in pheromonal responses have been documented in mammals ( Beny & Kimchi, 2014 ), insects have received far less attention, largely due to the assumption that their responses to pheromones are instinct-driven ( Dukas, 2008 ). However, social insects offer an excellent model to explore the flexibility of pheromonal responses. Their social organization creates strong asymmetries in fitness outcomes - where most individuals forego reproduction gaining only indirect fitness benefit, while a few gain direct and more substantial fitness benefits. This imbalance may favor the evolution of learning mechanisms that modify pheromonal responses and thus provides an ideal system to test the role of experience and learning in pheromone-guided behavior. 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Share A social bee can learn a novel queen pheromone Etya Amsalem , Abraham Hefetz bioRxiv 2025.05.30.657092; doi: https://doi.org/10.1101/2025.05.30.657092 Share This Article: Copy Citation Tools A social bee can learn a novel queen pheromone Etya Amsalem , Abraham Hefetz bioRxiv 2025.05.30.657092; doi: https://doi.org/10.1101/2025.05.30.657092 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Evolutionary Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41936) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15153) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)