Queen Pheromone Research Articles

Differences in honeybee queen pheromones revealed by LC-MS/MS: Reassessing the honest signal hypothesis

The honest signal hypothesis posits that social insect queens emit pheromonal signals that convey information about fecundity, which workers use to make decisions around investing in direct or indirect fitness. We used liquid chromatography-tandem mass spectrometry to measure honeybee (Apis mellifera) queen retinue pheromone (QRP) in relation to age, laying status, and acceptance using a protocol that enables concurrent metabolomic and lipidomic analyses. Older queens produced higher levels of the QRP components 9-R-hydroxydec-2(E)-enoic acid (9(R)-HDA), linolenic acid (LEA), and 4-hydroxy-3-methoxyphenylethanol (HVA) compared to younger queens, with HVA also correlating with ovary mass. However, ovary mass was not an influential metric for worker decision-making around queen acceptance; therefore, the relationship between HVA and ovary mass is merely an "honest signal" of a non-influential metric. Parallel metabolomic and lipidomic analyses showed that samples cluster according to queen age and mating status, but not ovary mass, revealing many other physiological changes occurring in the queen's early life.

The Evolution of Queen Pheromone Production and Detection in the Reproductive Division of Labor in Social Insect Colonies.

Structurally diverse queen pheromones and fertility signals regulate the reproductive division of labor of social insects, such as ants, termites, some bees, and some wasps. The independent evolution of sociality in these taxa allows for the exploration of how natural history differences in sender and receiver properties led to the evolution of these complex communication systems. While describing the different effects and the structural diversity of queen pheromones, we identify two major syndromes that mostly separate ants and wasps from bees and termites in their use of different pheromone classes. We compare olfactory receptor evolution among these groups and review physiological and hormonal links to fecundity and pheromone production. We explore the cases in which queen pheromone evolution is conserved, convergent, or parallel and those in which queen pheromone responses are more likely to be learned or innate. More mechanistic information about the pathways linking fecundity to queen pheromone production and perception could help close major knowledge gaps.

Early life exposure to queen mandibular pheromone mediates persistent transcriptional changes in the brain of honey bee foragers.

Behavioural regulation in insect societies remains a fundamental question in sociobiology. In hymenopteran societies, the queen plays a crucial role in regulating group behaviour by affecting individual behaviour and physiology through modulation of worker gene expression. Honey bee (Apis mellifera) queens signal their presence via queen mandibular pheromone (QMP). While QMP has been shown to influence behaviour and gene expression of young workers, we know little about how these changes translate in older workers. The effects of the queen pheromone could have prolonged molecular impacts on workers that depend on an early sensitive period. We demonstrate that removal of QMP impacts long-term gene expression in the brain and antennae in foragers that were treated early in life (1day post emergence), but not when treated later in life. Genes important for division of labour, learning, chemosensory perception and ageing were among those differentially expressed in the antennae and brain tissues, suggesting that QMP influences diverse physiological and behavioural processes in workers. Surprisingly, removal of QMP did not have an impact on foraging behaviour. Overall, our study suggests a sensitive period early in the life of workers, where the presence or absence of a queen has potentially life-long effects on transcriptional activity.

Innate and learned components of egg recognition in the ant Camponotus floridanus.

Insect societies discriminate against foreigners to avoid exploitation. In ants, helper workers only accept individuals with the familiar chemical cues of their colony. Similarly, unfamiliar eggs may get rejected at their first appearance in the nest. We investigated egg acceptance mechanisms by introducing different types of foreign eggs into worker groups of the ant Camponotus floridanus. Workers from established colonies familiar with queen-laid eggs always accepted eggs from highly fecund queens, but worker-laid eggs only after exposure for several weeks. Workers naive to eggs only rejected worker-laid eggs once they had prior exposure to eggs laid by highly fecund queens, suggesting that prior exposure to such eggs is necessary for discrimination. The general acceptance of eggs from highly fecund queens, irrespective of previous worker egg exposure, suggests an innate response to the queen pheromone these eggs carry. Workers learned to accept queen-laid eggs from different species, indicating high flexibility in learning egg-recognition cues. In incipient colonies with queen-laid eggs that carry a weak queen pheromone, worker-laid eggs were more likely to get accepted than queen-laid eggs from a different species, suggesting that the similarity of egg-recognition cues between the two types of C. floridanus eggs increases acceptance.

The genome of the blind bee louse fly reveals deep convergences with its social host and illuminates Drosophila origins

Social insects' nests harbor intruders known as inquilines,1 which are usually related to their hosts.2,3 However, distant non-social inquilines may also show convergences with their hosts,4,5 although the underlying genomic changes remain unclear. We analyzed the genome of the wingless and blind bee louse fly Braula coeca, an inquiline kleptoparasite of the western honey bee, Apis mellifera.6,7 Using large phylogenomic data, we confirmed recent accounts that the bee louse fly is a drosophilid8,9 and showed that it had likely evolved from a sap-breeder ancestor associated with honeydew and scale insects' wax. Unlike many parasites, the bee louse fly genome did not show significant erosion or strict reliance on an endosymbiont, likely due to a relatively recent age of inquilinism. However, we observed a horizontal transfer of a transposon and a striking parallel evolution in a set of gene families between the honey bee and the bee louse fly. Convergences included genes potentially involved in metabolism and immunity and the loss of nearly all bitter-tasting gustatory receptors, in agreement with life in a protective nest and a diet of honey, pollen, and beeswax. Vision and odorant receptor genes also exhibited rapid losses. Only genes whose orthologs in the closely related Drosophila melanogaster respond to honey bee pheromone components or floral aroma were retained, whereas the losses included orthologous receptors responsive to the anti-ovarian honey bee queen pheromones. Hence, deep genomic convergences can underlie major phenotypic transitions during the evolution of inquilinism between non-social parasites and their social hosts.

Developmental exposure to hormone-mimicking insect growth disruptors alters expression of endocrine-related genes in worker honey bee (Hymenoptera: Apidae) brains and hypopharyngeal glands.

Division of labor within a honey bee colony creates a codependence between bees performing different tasks. The most obvious example of this is between the reproductive queen and worker bees. Queen bees lay 1,000 or more eggs a day, while young worker bees tend and feed queens. Young workers and queens can be exposed to pesticides when foragers return to the hive with contaminated resources. Previous research has found negative effects of larval exposure to insect-growth disruptors (IGD) methoxyfenozide and pyriproxyfen, on adult responsiveness to artificial queen pheromone. The present work investigates potential physiological and molecular mechanisms underpinning this behavioral change by examining the development of hypopharyngeal glands and ovaries as well as the expression of genes related to reproduction and worker endocrine signaling in the brain and hypopharyngeal gland tissues. Though hypopharyngeal gland and ovary development were not altered by developmental exposure to IGDs, gene expression differed. Specifically, in the brain tissue, ilp1 was downregulated in bees exposed to pyriproxyfen during development, and Kr-h1 was downregulated in both methoxyfenozide- and pyriproxyfen-exposed bees. In the hypopharyngeal glands, Kr-h1, EcR-A, EcR-B, and E75 were upregulated in honey bees exposed to methoxyfenozide compared to those in the pyriproxyfen or control treatments. Here we discuss these results and their potential implications for the health and performance of honey bee colonies.

The use of drone congregation behaviour for population surveys of the honey bee Apis cerana

Honey bees (Apis spp.) are important pollinators in many natural and agro-ecosystems across the world. Effective means of surveying wild populations are therefore key to their conservation and management. One available survey method infers honey bee colony density from the genotype of drones (males) sampled from sites known as Drone Congregation Areas (DCAs). While this approach has been commonly used for the Western honey bee (A. mellifera), its feasibility for other Apis species is unknown. Here, we investigate drone congregation behaviour in the Asian honey bee Apis cerana in north-east Australia and its suitability for inferring colony density. As this A. cerana population is invasive, surveys in this case can aid in monitoring the population’s growth and spread. Over 5 years, we identified 30 DCAs, many of which were stable across time. DCAs were sheltered areas beside tree-lines or openings in the forest canopy. A. cerana drones joined DCAs during 1–2-h afternoon intervals and could be sampled at heights of 2–24 m via adhesive lines attached to helium balloons carrying lures coated in queen pheromone. Drones were more likely to be present at a DCA as temperature increased, though abiotic factors did not predict overall drone abundance. Drones could be sampled in low numbers even where colony density was extremely low. Based on the genotyping and inferred sibship of drones sampled at DCAs between 2016 and 2021, we estimate population density in Australia’s A. cerana to be in the range 1.1–8.1 colonies/km2. This extrapolates to a total population size in the range 11,000–83,000 colonies, with more refined estimates requiring better knowledge of drone flight distance and the effect of habitat on colony density. We conclude that population surveys based on drones from DCAs are possible for A. cerana and propose that this approach be part of a toolkit of methods used to monitor Asian honey bee populations in both their native and invasive ranges.

Chemical signatures of egg maternity and Dufour’s gland in Vespine wasps

Cuticular hydrocarbons (CHCs) are often used in the chemical communication among social insects. CHCs can be used in nestmate recognition and as queen pheromones, the latter allows the regulation of the reproductive division of labor. In the common wasp Vespula vulgaris, CHCs and egg-marking hydrocarbons are caste-specific, being hydrocarbon queen pheromones and egg maternity signals. Whether these compounds are conserved among other Vespinae wasps remains unknown. Queens, virgin queens, reproductive workers, and workers belonging to four different wasp species, Dolichovespula media, Dolichovespula saxonica, Vespa crabro, and Vespula germanica, were collected and studied. The cuticular hydrocarbons, egg surface, and Dufour's gland composition were characterized and it was found that chemical compounds are caste-specific in the four species. Quantitative and qualitative differences were detected in the cuticle, eggs, and Dufour's gland. Some specific hydrocarbons that were shown to be overproduced in the cuticle of queens were also present in higher quantities in queen-laid eggs and in their Dufour's gland. These hydrocarbons can be indicated as putative fertility signals that regulate the division of reproductive labor in these Vespine societies. Our results are in line with the literature for V. vulgaris and D. saxonica, in which hydrocarbons were shown to be conserved queen signals. This work presents correlative evidence that queen chemical compounds are found not only over the body surface of females but also in other sources, such as the Dufour's gland and eggs.

Evidence of queen-rearing suppression by mature queens in the little fire ant, Wasmannia auropunctata.

The little fire ant (LFA), Wasmannia auropunctata, is a serious invasive pest first reported on Hawaii Island in 1999, and has since spread and established itself across the island. LFA is considered one of the worst 100 invasive species and has significant ecological, agricultural, and public health impacts in invaded areas, which include much of the tropical New World. Although localized eradication efforts have proven successful, they are intensive and difficult to implement. Furthermore, LFA's high invasive-ability resists these control efforts in areas where the species is established and can re-infest treated areas. This research set out to determine whether LFA queens have a suppressant effect on new queen production in nests, as a first step in identifying a potential queen pheromone for LFA. A queen pheromone could offer a means to shutdown LFA reproductive capability, potentially by suppressing the production of new queens or inducing the execution of queens or queen-destined larvae. When queenless experimental nests and polygyne experimental nests were compared, six out of eight queenless nests successfully reared both new alate queens (2.25 queens/nest) and drones (3.63 drones/nest) to adulthood, whereas only three of eight polygyne nests reared sexual larvae that failed to develop to adulthood or even the pupal stage. These results suggest that dealate mature LFA queens suppress the production of new alate queens in LFA nests, and is the first evidence that LFA may utilize a queen pheromone.

Honeybee queen mandibular pheromone induces a starvation response in Drosophila melanogaster

Eusocial insect societies are defined by the reproductive division of labour, a social structure that is generally enforced by the reproductive dominant(s) or ‘queen(s)’. Reproductive dominance is maintained through behavioural dominance or production of queen pheromones, or a mixture of both.Queen mandibular pheromone (QMP) is a queen pheromone produced by queen honeybees (Apis mellifera) which represses reproduction in worker honeybees. How QMP acts to repress worker reproduction, the mechanisms by which this repression is induced, and how it has evolved this activity, remain poorly understood. Surprisingly, QMP is capable of repressing reproduction in non-target arthropods.Here we show that in Drosophila melanogaster QMP treatment mimics the starvation response, disrupting reproduction. QMP exposure induces an increase in food consumption and activation of checkpoints in the ovary that reduce fecundity and depresses insulin signalling. The magnitude of these effects is indistinguishable between QMP-treated and starved individuals. As QMP triggers a starvation response in an insect diverged from honeybees, we propose that QMP originally evolved by co-opting nutrition signalling pathways to regulate reproduction.

The effect of the brood and the queen on early gene expression in bumble bee workers' brains

Worker reproduction in social insects is often regulated by the queen, but can be regulated by the brood and nestmates, who may use different mechanisms to induce the same outcomes in subordinates. Analysis of brain gene expression patterns in bumble bee workers (Bombus impatiens) in response to the presence of the queen, the brood, both or neither, identified 18 differentially expressed genes, 17 of them are regulated by the queen and none are regulated by the brood. Overall, brain gene expression differences in workers were driven by the queen’s presence, despite recent studies showing that brood reduces worker egg laying and provides context to the queen pheromones. The queen affected important regulators of reproduction and brood care across insects, such as neuroparsin and vitellogenin, and a comparison with similar datasets in the honey bee and the clonal raider ant revealed that neuroparsin is differentially expressed in all species. These data emphasize the prominent role of the queen in regulating worker physiology and behavior. Genes that serve as key regulators of workers’ reproduction are likely to play an important role in the evolution of sociality.

Expression Profile and Ligand Screening of a Putative Odorant-Binding Protein, AcerOBP6, from the Asian Honeybee.

Simple SummaryThe olfactory sensillum, which is located in the antenna of insects, is the basic unit of the olfactory organ. Olfactory-related genes are expressed in the sensillum. It is believed that the process of olfaction recognition is mainly mediated by two gene families, odorant binding proteins (OBPs) and olfactory receptors (ORs). The honeybee possesses a large numbers of ORs, but few OBPs. Up to now, the function of OBPs in the honeybee has not yet been fully elucidated. In order to reveal the specific role of OBPs from Apis cerana cerana, we selected an OBP gene, AcerOBP6, which is highly expressed in the antennae of worker bees, acquired a purified protein via a prokaryotic expression system, and analyzed its function using bioinformatics, molecular biology, and electrophysiology. According to the result, AcerOBP6 was a protein with extensive binding affinity, and we speculated that its function was chiefly related to foraging. Overall, this research not only explains the essential role of OBPs in ligand binding, but also provides valuable resources to help researchers further understand the nature and mechanism of the olfactory system.Olfaction is essential in some behaviors of honeybee, such as nursing, foraging, attracting a mate, social communication, and kin recognition. OBPs (odorant binding proteins) play a key role in the first step of olfactory perception. Here, we focused on a classic OBP with a PBP-GOBP domain from the Asian honeybee, Apis cerana cerana. Beyond that, the mRNA expression profiles and the binding affinity of AcerOBP6 were researched. According to qRT-PCR analysis, AcerOBP6 transcripts were mainly expressed in the antennae of forager bees. In addition, we found that the expression level of AcerOBP6 was higher than that of AmelOBP6. The fluorescence competitive binding assay indicated that the AcerOBP6 protein had binding affinity with most of the tested odors, including queen pheromone, worker pheromone, and floral volatiles, among which the strongest one was linolenic acid (with a Ki value of 1.67). However, AcerOBP6 was not sensitive to the brood pheromones. A further study based on EAG assay revealed that the antennae had the strongest response to 2-heptanone. The EAG recording values of the selected ligands were all reduced after AcerOBP6 was silenced, with 8 of 14 declining significantly (p < 0.01) given that these odors could specifically bind to AcerOBP6. As revealed in our current study, AcerOBP6 might be a crucial protein involved in olfactory recognition for foraging. Overall, the research provides a foundation for exploring the olfactory mechanism of A. cerana cerana.

Queen Harems of Higher Termites are Regulated by Queen Pheromone

Colonies of higher termites (Termitidae) are usually headed by one king and one queen. Striking exceptions have recently been described in several species, whose founding queens prematurely die and are replaced by harems of up to hundreds of neotenic queens, arising from unfertilized eggs. This complex breeding system calls for the presence of a mechanism which would control the onset of the harem and its size. Here we show in one of such species, Embiratermes neotenicus, that the sesquiterpenoid (3R,6E)-nerolidol, emitted by queens and eggs, delays the development of new neotenic queens. We prove its effect as an airborne signal and demonstrate its olfactory detection. We conclude that (3R,6E)-nerolidol acts as a queen pheromone which may ensure the correct timing of the founding queen’s replacement and optimize the harem size according to colony population and fitness of reigning queen(s), being thus the key regulatory element of the harem breeding system.

Correlation of Royal Jelly Composition with Swarming Tendency in Honey Bees (Apis mellifera): A Review

Honey bees are eusocial insects which respond to warm weather, abundant food source by increasing their population through swarming to ensure the survival of the colony. To maintain a superior colony a queen must have a nutrient-rich diet and high egg production. Royal jelly is a high-quality food which has numerous beneficial properties required for proper growth, development, survival of the queen. Factors like congestion, lack of adequate queen pheromone, abnormal queen pheromone, pathogenic infections, exposure to pesticides influence the queen quality which further promotes non-reproductive swarming behaviour. Worker bees analyse the queen condition to prepare for supersedure or emergency queen rearing. This review paper highlights the influence of royal jelly composition on the queen quality, the impact of queen quality on swarming tendency, correlation between royal jelly composition and swarming tendency.

The Behavioral Toxicity of Insect Growth Disruptors on Apis mellifera Queen Care

As social insects, honey bees (Apis mellifera) rely on the coordinated performance of various behaviors to ensure that the needs of the colony are met. One of the most critical of these behaviors is the feeding and care of egg laying honey bee queens by non-fecund female worker attendants. These behaviors are crucial to honey bee reproduction and are known to be elicited by the queen’s pheromone blend. The degree to which workers respond to this blend can vary depending on their physiological status, but little is known regarding the impacts of developmental exposure to agrochemicals on this behavior. This work investigated how exposing workers during larval development to chronic sublethal doses of insect growth disruptors affected their development time, weight, longevity, and queen pheromone responsiveness as adult worker honey bees. Exposure to the juvenile hormone analog pyriproxyfen consistently shortened the duration of pupation, and pyriproxyfen and diflubenzuron inconsistently reduced the survivorship of adult bees. Finally, pyriproxyfen and methoxyfenozide treated bees were found to be less responsive to queen pheromone relative to other treatment groups. Here, we describe these results and discuss their possible physiological underpinnings as well as their potential impacts on honey bee reproduction and colony performance.

Bumble bee queen pheromones are context-dependent

Queen pheromones have long been studied as a major factor regulating reproductive division of labor in social insects. Hitherto, only a handful of queen pheromones were identified and their effects on workers have mostly been studied in isolation from the social context in which they operate. Our study examined the importance of behavioral and social context for the perception of queen semiochemicals by bumble bee workers. Our results indicate that a mature queen’s cuticular semiochemicals are capable of inhibiting worker reproduction only when accompanied by the queen’s visual presence and the offspring she produces, thus, when presented in realistic context. Queen’s chemistry, queen’s visual presence and presence of offspring all act to regulate worker reproduction, but none of these elements produces an inhibitory effect on its own. Our findings highlight the necessity to reconsider what constitutes a queen pheromone and suggest a new approach to the study of chemical ecology in social insects.

Pesticide Exposure During Development Does Not Affect the Larval Pheromones, Feeding Rates, or Morphology of Adult Honey Bee (Apis mellifera) Queens

Recent work demonstrated that honey bee (Apis mellifera L.) queens reared in pesticide-laden beeswax exhibit significant changes in the composition of the chemicals produced by their mandibular glands including those that comprise queen mandibular pheromone, which is a critical signal used in mating as well as queen tending behavior. For the present study, we hypothesized that pesticide exposure during development would alter other queen-produced chemicals, including brood pheromone in immature queens, thus resulting in differential feeding of queen larvae by nurse workers, ultimately impacting adult queen morphology. We tested these hypotheses by rearing queens in beeswax containing field-relevant concentrations of (1) a combination of tau-fluvalinate and coumaphos, (2) amitraz, or (3) a combination of chlorothalonil and chlorpyrifos. These pesticides are ubiquitous in most commercial beekeeping operations in North America. We observed nurse feeding rates of queen larvae grafted into pesticide-laden beeswax, analyzed the chemical composition of larval queen pheromones and measured morphological markers in adult queens. Neither the nurse feeding rates, nor the chemical profiles of immature queen pheromones, differed significantly between queens reared in pesticide-laden wax compared to queens reared in pesticide-free wax. Moreover, pesticide exposure during development did not cause virgin or mated adult queens to exhibit differences in morphological markers (i.e., body weight, head width, or thorax width). These results were unexpected given our previous research and indicate that future work is needed to fully understand how pesticide exposure during development affects honey bee queen physiology, as well as how various adult queen quality metrics relate to each other.

Cuticular hydrocarbons as cues of caste and sex in the German wasp Vespula germanica

The complex system of communication used by social insects is responsible for their success on Earth. Cuticular hydrocarbons (CHCs) are considered the most commonly used form of communication among social insects. The best-known function of CHCs is to distinguish nestmates, providing cues to the whole colony to identify potential threats. In some species, CHCs function as queen pheromones, which contribute to the queens’ ability to regulate the reproductive division of labor. Additionally, differences of CHCs were described in castes and they are also important in sex recognition. Here, we reinvestigated the chemical composition on the cuticle of German wasp Vespula germanica and we found that proportions of linear alkanes were more abundant in the cuticle of queens than other castes. Chemical analysis between caste and sex, including data of virgin queens and males, showed that each group possesses different compositions on their cuticle, being mostly quantitative differences. Thus, individuals of V. germanica may recognize and discriminate caste and sex through subtle chemical variations, or alternatively, based on templates used in nestmate recognition. The chemical analysis provides correlative evidence of CHCs being used as cues in the chemical communication of this species.

Hormonal modulation of reproduction and fertility signaling in polistine wasps

In social insects, it has been suggested that reproduction and the production of particular fertility-linked cuticular hydrocarbons (CHC) may be under shared juvenile hormone (JH) control, and this could have been key in predisposing such cues to later evolve into full-fledged queen pheromone signals. However, to date, only few studies have experimentally tested this “hormonal pleiotropy” hypothesis. Here, we formally test this hypothesis using data from four species of Polistine wasps, Polistes dominula, Polistes satan, Mischocyttarus metathoracicus, and Mischocyttarus cassununga, and experimental treatments with JH using the JH analogue methoprene and the anti-JH precocene. In line with reproduction being under JH control, our results show that across these four species, precocene significantly decreased ovary development when compared with both the acetone solvent-only control and the methoprene treatment. Consistent with the hormonal pleiotropy hypothesis, these effects on reproduction were further matched by subtle shifts in the CHC profiles, with univariate analyses showing that in P. dominula and P. satan the abundance of particular linear alkanes and mono-methylated alkanes were affected by ovary development and our hormonal treatments. The results indicate that in primitively eusocial wasps, and particularly in Polistes, reproduction and the production of some CHC cues are under joint JH control. We suggest that pleiotropic links between reproduction and the production of such hydrocarbon cues have been key enablers for the origin of true fertility and queen signals in more derived, advanced eusocial insects.

Cuticular and Dufour's Gland Chemistry Reflect Reproductive and Social State in the Facultatively Eusocial Sweat Bee Megalopta genalis (Hymenoptera: Halictidae).

Queen pheromones evolved independently in multiple eusocial insect lineages, in which they mediate reproductive conflict by inhibiting worker ovarian development. Although fundamentally important for reproductive division of labor - the hallmark of eusociality - their evolutionary origins are enigmatic. Here, we analyze cuticular and Dufour's gland chemistries across alternative social and reproductive phenotypes in Megalopta genalis bees (tribe Augochlorini, family Halictidae) that facultatively express simple eusociality. Reproductive bees have distinct overall glandular and cuticular chemical phenotypes compared with non-reproductive workers. On the cuticle, a likely site of signal transmission, reproductives are enriched for certain alkenes, most linear alkanes, and are heavily enriched for all methyl-branched alkanes. Chemicals belonging to these compound classes are known to function as fertility signals in other eusocial insect taxa. Some macrocyclic lactones, compounds that serve as queen pheromones in the other eusocial halictid tribe (Halictini), are also enriched among reproductives relative to workers. The intra-population facultative eusociality of M. genalis permits direct comparisons between individuals expressing alternative reproductive phenotypes - females that reproduce alone (solitary reproductives) and social queens - to highlight traits in the latter that may be important mediators of eusociality. Compared with solitary reproductives, the cuticular chemistries of queens are more strongly differentiated from those of workers, and furthermore are especially enriched for methyl-branched alkanes. Determining the pheromonal function(s) and information content of the candidate signaling compounds we identify will help illuminate the early evolutionary history of queen pheromones, chemical signals central to the organization of insect eusocial behavior.