Bumblebees do not prefer consistent floral scents over variable ones

Bumblebees do not prefer consistent floral scents over variable ones | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bumblebees do not prefer consistent floral scents over variable ones Mélissa Armand, Lisa Zeilmann, Christian Weinzettl, Tomer J. Czaczkes This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6292403/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 5 You are reading this latest preprint version Abstract To attract pollinators, flowering plants evolve diverse sensory traits into compelling signals. Floral scent, in particular, plays a key role in drawing bees from a distance and shaping their foraging choices. Scent composition varies widely, including across flowers of the same plant species. Yet, it is unclear whether scent variability influences bee flower choices, and thus whether plants would be under selection to minimise variation in their scent composition. Since bees typically avoid variability in rewards, we hypothesised they would favour flowers with more consistent scents. To test this, we trained individual bumblebees ( Bombus terrestris ) on two equally rewarding flower arrays: one with a consistent scent blend across flowers, and the other with variable scent blends between flowers. Contrary to expectations, bees showed no preference for scent consistency. They readily foraged from both arrays across bouts and did not favour either flower type in the binary choice test. To the best of our knowledge, this is the first study to examine how bees respond to scent variability. A better understanding of scent profile preferences in pollinators could offer new insights into their co-evolution with plants and the development of floral traits. More broadly, further research is needed on how pollinators respond to variability and unpredictability in neutral cues like scent or colour, a largely overlooked aspect of foraging decision-making. Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Most flowering plants depend on pollinators for reproduction (Klein et al., 2007), so their flowers act like billboards, using various colours, shapes, and scents to attract them (Dobson, 1994 ). Floral scents, especially, play a key role in pollination (Burkle et al., 2020 ): scent bouquets attract bees from far away (Raguso, 2004 ) and often serve as bees’ primary cue for deciding whether to land on a flower (Kunze et al., 2001; Raguso, 2008a ; Sprayberry, 2018 ). Yet, despite their influence on bees’ foraging decisions (Farré-Armengol et al., 2015; Larue et al., 2016 ), the role of scent in flower choices remains understudied in comparison to flower colours and shapes (Fenster et al., 2004 ; Raguso, 2008b ). Floral scents consist of complex blends of volatile organic compounds (Knudsen et al., 2006 ), released from various parts of the flower (Pichersky et al., 1994 ; Raguso and Pichersky, 1999 ). These scent bouquets act as unique floral identifiers (Raguso, 2008a ) and are highly diverse across plant species (Raguso and Pichersky, 1999 ; Levin et al., 2003 ). Scent composition can also vary across plant populations and even among individual flowers within the same species (Raguso et al., 2003 ; Burdon et al., 2015 ; Delle-Vedove et al., 2017 ). However, the causes of intraspecific scent variation remain poorly studied and understood (Majetic et al., 2009 ; Raguso, 2020 ), as well as its effects on pollinator foraging behaviour. Scent cues allow flower-visiting insects to associate flowers with pollen and nectar rewards (Kevan and Baker, 1983 ; Wells and Wells, 1985 ; Giurfa, 2007 ), and help them distinguish between plant species (Dobson, 1994 ). Both honeybees and bumblebees can detect differences in floral scent blends (Laloi and Pham-Delègue, 2004 ; Wright et al., 2005 ), and small variations in compound ratios were shown to strongly affect their flower choices (Dobson, 1994 ; Raguso, 2004 ; Tan and Nishida, 2012 ; Solís-Montero et al., 2018 ). Like floral colours, bees have innate preferences for certain floral volatiles or scent blends (Raguso, 2008a ; Schiestl and Dötterl, 2012 ). However, it is unclear whether bees also prefer certain patterns of scent variability. Floral scent is a highly variable trait that plants can adjust to reflect their current state (Dudareva et al., 1996 ; Dobson, 1994 ). For instance, scented nectar can inform pollinators about reward availability (Heinrich, 1979 ; Raguso, 2004 ; Gervasi and Schiestl, 2017). Plants can also modify their scents to better attract pollinators (Dudareva et al., 2004 ; Raguso, 2008a ; Leonard et al., 2011 ). For example, some flowers adjust the intensity of their scent emissions throughout the day to match pollinator activity (Loughrin et al., 1990 ; Raguso, 2008b ; Wright and Thomson, 2005 ). Scent composition may also vary at different stages of flower development (Majetic et al., 2015; Burkle et al., 2020 ). Do bees favour plant species with more consistent or variable scent profiles across flowers? Pollinators not only respond to floral traits but also influence how these traits evolve through selection pressure, including scent (Parachnowitsch et al., 2013 ; Schiestl and Johnson, 2013 ; Ollerton et al., 2011 ). Stabilising selection driven by pollinator preferences is well-established for flower colour (Goulson, 1999 ; Whibley et al., 2006 ), and similar selection may also shape scent composition (Huber et al., 2005 ; Mant et al., 2005 ; Salzmann et al., 2007 ). The pollination syndrome hypothesis suggests that unrelated plant species visited by similar pollinators tend to develop similar floral traits, such as scent composition (Fenster et al., 2004 ; Dobson, 2006 ; Farré-Armengol et al., 2020 ), although this idea remains debated (Rosas-Guerrero et al., 2014; Ollerton et al., 2015). Bumblebees tend to specialise and forage on a few flower species (Heinrich, 1979 ; Chittka et al., 1999 ). Such flower constancy benefits plants by ensuring that bees repeatedly visit the same species, increasing pollination efficiency (Heinrich, 1977 ; Waser, 1986 ). Like colours, scents promote flower constancy in pollinators (Gegear and Laverty, 2005 ; Gegear, 2005 ). In turn, this flower constancy probably contributes to stabilising floral traits, including scent profiles (Chittka et al., 1999 ; Goulson, 1999 ). This tendency to be flower constant may reflect a broader preference for consistency in bee foraging decisions. In fact, bees typically exhibit an aversion to variability and unpredictability in rewards (see review by Anselme, 2018 ). Similarly, we propose that bees may favour plant species with more consistent scent profiles across flowers, potentially placing selection pressure on plants to tightly control their scent composition. Here, we investigated whether bumblebees ( Bombus terrestris ) prefer flowers with either consistent or variable scents. To test this, we trained individual bees on two equally rewarding arrays of artificial flowers: all yellow or all green. In one array, all flowers contained sucrose solution scented with a fixed ratio of two artificial food flavourings, creating a consistent scent bouquet. In the other array, flowers had varying ratios of a different pair of scents, making varying, unpredictable scent bouquets. We hypothesized that bees would favour the consistent scent flower, being more predictable than the other flower. We predicted that in a binary choice test, bees would first visit the flower colour associated with the consistent scent. MATERIAL AND METHODS Colony setup Commercial Bombus terrestris colonies were purchased from Koppert (The Netherlands) and kept under controlled laboratory conditions at 22–24°C with a 14:10 light:dark cycle. Colonies were housed in wooden nestboxes, each connected to its respective flight arena (60 × 50 × 35 cm) leading to a small chamber (6 × 5 × 3 cm). The chamber featured a second tube that provided direct access to the arena and was fitted with transparent, removable shutters to regulate bee movement between the nest and arena (see apparatus Fig. 1 ). Bees were provided daily with pollen balls made from a mix of organic flower pollen pellets and 35% (w/w) sucrose solution, placed directly in the nestboxes. During the day, workers foraged freely on artificial flowers in the flight arena, which offered 35% (w/w) sucrose solution and were regularly refilled. Active foragers were captured and marked on the thorax with uniquely numbered, coloured tags, and considered for selection in the experiment on the same day. A total of 48 bees participated in the experiment from five colonies in May–June 2022 (see colony details in Supplement S1 ). Artificial flowers Each artificial flower consisted of a transparent plastic cup with a white lid (height: 4.5 cm, diameter: 3.8 cm), topped with a 2 mm-thick disc of coloured rubber foam. At the centre, a small opaque white resin cup (diameter: 4 mm, depth: 6 mm) was inserted into a pre-cut hole and filled with sucrose solution (Fig. 1 ). In the experiment, flowers were either yellow (peak reflectance: ~520–700 nm) or green (peak reflectance: ~530 nm; see Supplement S2 for reflectance curves). We selected these colours for their perceptual similarity while still allowing bees to differentiate them (see control experiment below), and to avoid strong innate preferences typically observed for blue or violet flowers (Gumbert, 2000 ; Raine and Chittka, 2007 ). Between experimental sessions, bees were pre-trained on bicoloured, half green half yellow flowers, to ensure they associated sucrose rewards equally with both flower colors used in the experiment (Raine and Chittka, 2008 ). During training, bees foraged on an array of flowers consisting of a 3 × 3 grid of either all-yellow or all-green flowers. In the binary choice test, the array contained 5 green and 5 yellow flowers, arranged in two front rows of three flowers and a third row of four flowers (see Fig. 1 below). Flowers were spaced 3.5 cm apart and mounted on a grey-painted plate matching the all-grey arena. The plate ensured consistent flower placement and allowed for easy flower replacement between foraging bouts. Scented sucrose solutions Artificial flowers provided 35% w/w sucrose solution at a fixed volume, determined individually for each bee based on an estimate of its honey crop size (see below). Sucrose solutions were scented using strawberry, rose, lemon, or vanilla food flavourings (Seeger, Springe, Germany). We created six artificial scent bouquets by mixing the flavourings in different combinations: strawberry–rose, lemon–vanilla, strawberry–vanilla, lemon–rose, strawberry–lemon, and vanilla–rose. Each combination was prepared in three different ratios: 1:1 (equal parts of both scents), 1:3 (25% scent A, 75% scent B), or 3:1 (75% scent A, 25% scent B). Bees were familiarised with each scent (strawberry, rose, lemon and vanilla) 48 hours before the experiment, by placing filter papers soaked with 25 µL of each food flavouring in the four corners of the nestboxes, allowing airborne scents to disperse. This ensured that bees were not reluctant to collect sucrose solution during the experiment due to unfamiliar scents. Estimation of crop size To determine the appropriate volume of sucrose solution used per flower in the experiment, we first estimated the crop capacity of individual bees. Bee foraged on a 3 × 3 array of bicoloured flowers (half green, half yellow), with each flower providing 15 µL of a 35% w/w sucrose solution. We recorded the number of flowers collected over two consecutive foraging bouts and estimated the crop size of the bees by averaging the total volume collected across both bouts. This average volume was then divided by nine, and the resulting volume was used as the reward per flower during the experiment. This ensured that the bees could collect all available rewards in each foraging bout. Training After its crop size was estimated, each bee was trained for six consecutive foraging bouts on two different 3 × 3 flower arrays, alternating between them in each bout. Each flower array was associated with a flower colour (green or yellow) and a scent combination (strawberry–rose, lemon–vanilla, strawberry–vanilla, lemon–rose, strawberry–lemon, or vanilla–rose). Bees experienced a distinct scent combination for each flower colour; for example, if one array had green flowers scented with strawberry–lemon, the other had yellow flowers scented with the remaining two scents (e.g., rose–vanilla). We assigned different scent combinations to each flower colour to ensure clear differentiation between flower types and allowed the use of three different scent ratios (1:1, 1:3, and 3:1) in the variable flower type, while maintaining a consistent 1:1 ratio in the consistent flower (see below). The flower arrays were one of two types: Consistent flower array : All flowers in the array had the same scent combination at a fixed 1:1 ratio (equal parts of both scents); Variable flower array : Flowers in the array had varying scent ratios: some 1:1, some 1:3 (25% scent A, 75% scent B), and some 3:1 (75% scent A, 25% scent B). For example, if a bee experienced green flowers as the consistent array, all green flowers would have strawberry–lemon in a fixed 1:1 ratio, and the yellow flowers in the variable array would contain rose–vanilla with varying scent ratios (1:1, 1:3, and 3:1). Half of the bees experienced the consistent flower array as green and the variable array as yellow (n = 24 bees), and the other half the reverse (n = 24 bees). Bees were required to consistently collect sucrose solution from both array types to ensure they perceived each scent combination as an acceptable reward. Flowers were replaced with clean ones each new bout to prevent scent marks from influencing subsequent foraging (Goulson et al., 2000 ; Saleh et al., 2007 ). Binary choice test After the six training bouts, each bee was presented with an array of 10 flowers — 5 yellow and 5 green, arranged so that neighbouring flowers alternated in colour. The flowers were unrewarded and filled with unscented plain water. Bees were randomly assigned one of two array layouts, where the front row contained either two green flowers and one yellow (“green-biased” layout), or the reverse (“yellow-biased” layout). We recorded the first flower choices as an indicator of preference. Control experiment A pilot was conducted before the main experiment to test whether bees could differentiate between green and yellow flowers. The setup mirrored the main experiment, where individual bees completed six training bouts to a 3 × 3 flower array of a single colour and alternating between arrays each bout, followed by a binary choice test with both flower colours. Instead of being scented, one flower type offered 25 µL of a low-quality sucrose solution (15% w/w), while the other provided 35% w/w sucrose solution. This clear difference in reward quality allowed us to assess whether bees could correctly identify the flower colour associated with the higher-quality reward. All tested bees (n = 10) successfully chose first the flower colour associated with high-quality reward in the final test. Data analysis The binary choice test was video-recorded for each bee with a camera (Sony HDR-CX220) positioned above the flight arena. Bee behaviour was analysed using the event-logging software BORIS (v8.6). We tested the hypothesis that bees would favour the flower colour (green or yellow) associated with a consistent scent. To assess this, we recorded (1) the first flower visit and (2) the first 10 flower visits during the binary choice test, as indicators of preference. For the first flower visit, we built a generalized linear mixed model (GLMM) with a binomial distribution, using first flower scent choice (1 for consistent, 0 for variable) as the response variable and flower colour (green or yellow) as the predictor. Random effects included bee colony, first flower scent encountered (i.e., whether the first training bout involved the consistent or variable array), first flower colour encountered (green or yellow in the first training bout), and binary test layout (“green-biased” or “yellow-biased”). Predicted probabilities of first choice were assessed through post hoc pairwise comparisons with a Tukey correction. For the first ten flower visits, we fitted a GLMM with a binomial distribution, using flower scent choices (1 for consistent, 0 for variable) as the response variable and flower colour (green or yellow) as the predictor. Random effects included individual bee identities nested within their colony, first flower scent encountered, first flower colour encountered and binary test layout. Predicted probabilities of flower choices were evaluated through post hoc pairwise comparisons with a Tukey correction. Data processing was conducted in Python (v3.11, Python Software Foundation, 2023) using the pandas library (McKinney, 2010 ) for data structuring and seaborn (Waskom, 2021) and Matplotlib (Hunter, 2007 ) for data visualization. Statistical analyses were performed in R (v4.1, R Core Team, 2022) using the glmmTMB package (Brooks et al., 2017 ) for GLMMs, and emmeans (Lenth, 2020 ) for post hoc tests. Model residuals were evaluated with the DHARMa package (Hartig, 2020 ). Complete statistical analyses and datasets are available on Zenodo ( https://doi.org/10.5281/zenodo.14993082 ). RESULTS First flower choice 31 of the 48 tested bees (64.6% ± 8.6%) first visited a yellow flower in the binary choice test, while 17 bees (35.4% ± 11.6%) chose a green flower. This difference was borderline significant ( p = 0.059, two-tailed exact binomial test, 95% CI: 49.5–77.8%). Regarding flower scent choice, 27 bees (56.3% ± 9.5%) chose a flower associated with variable scents, and 21 bees (43.7% ± 10.8%) a flower associated with a consistent scent. We then tested the combined effects of flower scent, colour, and random effects on bees’ first flower choice (see model (1) in Data analysis). Bees showed no preference between flowers associated with consistent or variable scents (GLMM, binomial family; intercept: Estimate = -0.36 ± 0.50, z = -0.72, p = 0.47; N = 48; Fig. 2 A), and flower colour had no significant effect on this choice (X² = 0.073, df = 1, p = 0.79). First 10 flower visits During their first ten visits in the binary choice test, bees visited yellow flowers 51.2% (± 3.3%) of the time and green flowers 48.8% (± 3.4%). Regarding flower scent choice, 50.3% (± 3.3%) of visits were to flowers associated with variable scents, and 49.7% (± 3.3%) to flowers associated with a consistent scent. The GLMM (see model (2) in Data Analysis) revealed no preference between flowers associated with consistent or variable scents (binomial family; intercept: Estimate = -0.007 ± 0.14, z = -0.05, p = 0.96; N = 457; Fig. 2 B), and flower colour had no effect on first 10 visits (χ² = 0.004, df = 1, p = 0.95). Scents preferences Overall, bees showed no preference for any scent bouquet in the binary choice test (Chi-square goodness-of-fit test: X² = 1.75, df = 5, p = 0.88; Fig. 3 A) and did not favour any individual scent—lemon, rose, vanilla, or strawberry (X² = 1.08, df = 3, p = 0.78; Fig. 3 B). DISCUSSION We tested whether bees had a preference between two equally rewarding flower arrays, yellow or green, one with consistent scents across flowers and the other with variables scents between flowers. Contrary to expectations, bees showed no preference for flowers with a consistent scent. During training, they readily collected sucrose solution flavoured with varying scent combinations and ratios, and in the binary choice test, they did not favour the flower colour associated with a consistent scent. To our knowledge, this is the first study to explicitly test whether bees prefer consistent or variable scents—or any other neutral cue like colour or shape—independently of reward variation. Every tested bee collected all sucrose rewards in both flower arrays, regardless of scent composition. This reflects bees' ability to rapidly associate scents with rewards (Kunze and Gumbert, 2001 ; Wright and Schiestl, 2009 ; Giurfa, 2007 ), even when they have innate preferences for specific scent volatiles (Milet-Pinheiro et al., 2013 ; Raguso, 2008a ). Surprisingly, bees visited and accepted every flower in the variable array, despite previous findings that bumblebees exhibit even stronger flower constancy when flowers differ in multiple sensory cues, such as scents and colours (Wells and Wells, 1985 ; Gegear and Laverty, 2001; Gegear, 2005 ). However, recent research has challenged the idea of high flower constancy in bumblebees; Yourstone et al. ( 2023 ) found that bees were less flower constant than expected, with only 23% of their foraging trips occurring on the same flower species. In the binary choice test, bees showed a slight tendency to visit yellow flowers first, though this preference was not significant. Bees are known to have an innate preference for yellow flowers (Lunau, 1990 ; Gumbert, 2000 ). To ensure that choices were based on learned associations, we used unscented, unrewarded flowers in the binary test. Hence it is unclear whether scent preferences would have outweighed colour preferences if both cues were present simultaneously. Notably, Larue et al. (2015) found that floral scent had a stronger influence than visual traits in attracting flower-visiting insects. Were bees in our experiment able to distinguish between scent combinations, particularly the different scent blend ratios in the variable flower array? Studies show that bees can detect individual volatiles within complex scent blends, influencing their flower choices (Laloi and Pham-Delègue, 2004 ; Locatelli et al., 2016 ), and honeybees can even perceive subtle differences in the ratio of two scents (Wright et al., 2005 ; Bateson et al., 2011 ). Moreover, since our flowers contained scented sucrose solution, bees could also taste them; Robertson ( 2019 ) suggests that olfactory and gustatory receptors may overlap in bumblebees. In nature, scented nectar contains volatile organic compounds, which pollinators use to assess reward availability before landing on a flower (Heinrich, 1979 ; Raguso, 2008b ), and can impact their foraging behaviour (Raguso, 2004 ). Given this, it seems likely that bees could sense the variability in the variable flowers, and distinguish this from the fixed-ratio flowers. Although no study has directly tested bees’ preference for consistency in neutral cues, some have examined their responses to inconsistent rewarding cues. Honeybees tended to avoid choices where uncertain visual cues predict reward or punishment (Perry and Barron, 2013 ). Andrew et al. ( 2014 ) found that honeybees trained to discriminate between a rewarding and a punishing scent blend preferred a novel scent that was more distinct from the punishing one, rather than similar to the reward. Likewise, Lynn et al. ( 2005 ) showed that bumblebees favoured novel colours that minimised the risk of choosing an unrewarding flower. This phenomenon, known as “peak shift” (Hanson, 1959 ), occurs when animals develop a preference for a more extreme version of a rewarded stimulus to avoid similar, non-rewarded ones. These findings suggest that bees may prefer consistent cues over variable ones, to reduce uncertainty. Stress may further amplify this avoidance of inconsistent cues: Bateson et al. ( 2011 ) found that shaken honeybees were less likely to respond to ambiguous scents associated with rewards. When it comes to rewards preferences, bees are generally risk-averse and tend to avoid variability (see review by Anselme, 2018 ). They typically prefer consistent nectar amounts over variable ones (Real, 1981 ; Waddington et al., 1981 ; Shafir et al., 1999 ), which is likely in part due to the psychophysics of reward perception (Kacelnik and Bateson, 1996 ). When resources are variable, bumblebees also forage less efficiently (Dunlap et al., 2017 ) and rely more on social information for flower choice (Smolla et al., 2016 ). However, their sensitivity to variability depends on context. In some contexts, bees showed indifference to fluctuations in nectar concentration or volume (Waddington, 1995 löp and Menzel, 2000 ) and when variable distribution did not include null rewards (Drezner-Levy and Shafir, 2007 ). They may even favour variable rewards when advantageous: For example, bumblebees initially preferred consistent rewards but shifted to variable ones when colony nectar reserves were low, both in natural foraging conditions (Cartar, 1991 ) and in laboratory settings (Cartar and Dill, 1990). Bees' preferences for specific floral volatiles are well-documented (Knudsen et al., 2006 ; Farré-Armengol et al., 2017 ; Benelli et al., 2017 ; Bisrat and Jung, 2022 ), and they readily associate these scents with rewards (Wells and Wells, 1985 ; Wright and Schiestl, 2009 ; Giurfa, 2007 ). Bees can even retain scent memories longer than visual cues (Menzel, 1993 ; Kunze and Gumbert, 2001 ). Bumblebees, in particular, use floral scents as social cues, with foragers transferring scent compounds within the nest to inform nestmates (Molet et al., 2009 ). They also favour flowers that match the scents collected by successful foragers (Dornhaus and Chittka, 1999 ). However, bees learn consistent scents more effectively than variable ones (Wright and Thomson, 2005 ; Wright and Schiestl., 2009). For instance, Wright et al. ( 2008 ) found that honeybees reject scent-modified flowers even if they contain familiar scent compounds, highlighting the importance of scent consistency for pollinator recognition. Many rewardless flowers emit weak or highly variable scent, likely to avoid detection by scent-learning pollinators (Jersáková and Johnson, 2006 ; Salzmann et al., 2007 ). Salzmann et al. ( 2007 ) found that rewarding orchids produce strong, consistent scents that bees can detect, whereas deceptive orchids emit weak, highly variable scents. Similarly, floral compounds that attract pollinators tend to be more consistent across populations and species, while non-attractive compounds show greater variability (Mant et al., 2005 ; Huber et al., 2005 ). Nonetheless, floral scent remains a highly variable trait (Dudareva et al., 1996 ), even among individual flowers within the same species (Burdon et al., 2015 ). While plants adjust scent emissions to attract pollinators (Majetic et al., 2015; Burkle et al., 2020 ), scent bouquets are also influenced by environmental factors (Dudareva et al., 1999; Raguso, 2008b ). Studies suggest that intraspecific variation in floral scent may help attract local pollinators (Soler et al., 2011 ; Larue et al., 2016 ; Vega-Polanco et al., 2023 ). Fülöp and Menzel ( 2000 ) propose that bees’ tolerance for scent variability may be an adaptation to cope with unreliable floral resources. Floral scents, including scented nectar, likely serve as honest signals to pollinators (Howell and Alarcón, 2007 ; Gervasi and Schiestl, 2017). Bees often select flowers based on their scent composition (Pichersky and Raguso, 2018 ; Knudsen and Gershenzon, 2020 ) or the intensity of scent emissions (Majetic et al., 2009 ; Parachnowitsch et al., 2013 ), which inform foragers about nectar and pollen availability. We suggest that bees are likely to respond to olfactory cues regardless of whether they are more variable or consistent, as long as they can reliably associate them with rewards. Our findings highlight bees' ability to rapidly learn scent-reward associations and show that they forage equally on flower arrays with both variable and consistent scents, and show no preference between them. This in turn suggests that plants may not be under strong selection to reduce inter-flower scent variability, at least in terms compound ratios. Maintaining tight control over a trait in the face of environmental variation is costly, and if no selection for this maintenance is present, we would expect such traits to fluctuate, as indeed is the case with intraspecific scent variation between flowers (Burdon et al., 2015 ; Delle-Vedove et al., 2017 ). However, our controlled experiment likely oversimplified the complex sensory landscape bees navigate in nature, and further research is needed to understand how they respond to scent variability within flower patches. Despite its importance, the role of floral scent in bee foraging has been largely overlooked (Fenster et al., 2004 ; Raguso, 2004 , 2008). More generally, future studies should examine whether bees' aversion to variability in rewards extends to other neutral cues like morphology or colour, as the foraging preferences of plant pollinators play a key role in shaping floral trait evolution (Ollerton et al., 2011 ). Declarations SUPPLEMENTARY MATERIAL S1 contains the dataset of the main experiment, S2 provides the reflectance curves of the artificial flowers, and S3 the statistical analysis of the experiment. All supplementary materials are available on Zenodo (https://doi.org/10.5281/zenodo.14993082). ACKNOWLEDGMENTS We want to thank A. Koch and M. Kietniz for their assistance with data collection, and K. Hartmannsgruber for video analysis. Special thanks to A. Avarguès-Weber for providing spectrometer measurements of the artificial flowers. FUNDING M. A. was supported by an ERC Starting Grant to T. J. C. [H2020-EU.1.1. #948181] and T. J. C. was supported by a Heisenberg Fellowship from the Deutsche Forschungsgemeinschaft [#462101190]. AUTHORS’ CONTRIBUTION Mélissa Armand: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Writing - Original Draft, Writing - Review and Editing, Visualization. Lisa Zeilmann: Investigation. Christian Weinzettl : Investigation. Tomer J. Czaczkes: Conceptualization, Methodology, Validation, Resources, Writing - Review and Editing, Supervision, Project administration, Funding acquisition. CONFLICTS OF INTERESTS The authors declare they have no conflicts of interest in the production of this study. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6292403","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436916768,"identity":"e19fbee3-8ee2-4000-b48e-48e49853534c","order_by":0,"name":"Mélissa Armand","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYNACNhDBfABISMiQooUtAaSFhxQtPAZgkqBi/tmnEz8XlDHY87ef+fzqRo0FDwP74aMb8GmROJe7WXrGOYbEGWdyt1nnHAM6jCct7QZea87wbpDmbWNIYDiQu804hw2oRYLHDK8W+TO8m38DtdjLn3/zzDjnHxFaDM7wbgPZwrjhRg7z49w2IrQYArVY85yTSNx445kZc26fBA8bIb/IAR12m6fMxl7ufPLjzznf6uT42Q8fw+99CJAAEWwQkgjlcMD8gRTVo2AUjIJRMHIAAH5xQtgHBsCpAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3190-5852","institution":"Universitat Regensburg","correspondingAuthor":true,"prefix":"","firstName":"Mélissa","middleName":"","lastName":"Armand","suffix":""},{"id":436916769,"identity":"ed23f8ce-f97a-4ec3-b483-921352a77743","order_by":1,"name":"Lisa Zeilmann","email":"","orcid":"","institution":"Universitat Regensburg","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"Zeilmann","suffix":""},{"id":436916770,"identity":"5247db84-b982-40f5-a041-cb157f24dd0e","order_by":2,"name":"Christian Weinzettl","email":"","orcid":"","institution":"Universitat Regensburg","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Weinzettl","suffix":""},{"id":436916771,"identity":"d2d49ec3-c7de-4830-8563-9e850c40a547","order_by":3,"name":"Tomer J. Czaczkes","email":"","orcid":"","institution":"Universitat Regensburg","correspondingAuthor":false,"prefix":"","firstName":"Tomer","middleName":"J.","lastName":"Czaczkes","suffix":""}],"badges":[],"createdAt":"2025-03-24 06:58:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6292403/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6292403/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81090260,"identity":"8e2bfbff-9dc9-4287-aaad-51a64c563e97","added_by":"auto","created_at":"2025-04-22 07:02:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":603092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTop view of the experimental setup, to scale.\u003c/strong\u003eThe 3D model, created in Blender, illustrates a binary choice bout with a mixed flower array of yellow and green flowers, arranged in a “yellow biased” layout (\u003cem\u003ei.e.\u003c/em\u003e, two yellow flowers on the front row).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6292403/v1/deda953d926101a4c11b070c.png"},{"id":81091375,"identity":"ddbff0c4-29ff-4f74-8852-3bd9dce23f7b","added_by":"auto","created_at":"2025-04-22 07:10:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":130516,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstimated probabilities of choosing the consistent scent flower for each flower colour in the binary choice test. A)\u003c/strong\u003e Proportion of choices in the first flower visit (N = 48 visits); \u003cstrong\u003eB)\u003c/strong\u003e Proportion of choices across the first 10 flower visits (N = 457 visits). Probabilities were derived from the GLMM and calculated through post hoc pairwise comparisons. The dotted red line represents a 50% chance of choosing either flower type. Error bars represent the standard errors of the predictions, and statistical differences within treatments are indicated as \u003cstrong\u003en.s.\u003c/strong\u003e (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6292403/v1/b8269020ab06151791daf1b0.png"},{"id":81090258,"identity":"704e0858-3991-4944-be2e-92d46bc13a27","added_by":"auto","created_at":"2025-04-22 07:02:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProbabilities of choosing each flower scent in the binary choice test.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Proportion of choices for each scent bouquet (N = 48 visits): lemon–rose (LR), lemon–vanilla (LV), rose–vanilla (RV), strawberry–lemon (SL), strawberry–rose (SR), or strawberry–vanilla (SV). The dotted line represents the expected random choice (16.67%). \u003cstrong\u003e(B)\u003c/strong\u003e Proportion of choices for each individual scent (N = 48 visits): Lemon (L), Rose (R), Vanilla (V), or Strawberry (S), with the dotted red line indicating a 50% chance of choosing either scent. Error bars represent standard errors, and statistical differences between scents are indicated as \u003cstrong\u003en.s.\u003c/strong\u003e (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6292403/v1/caaf0373541fed1b66b0db91.png"},{"id":81092013,"identity":"f991eb38-3fc1-497f-9b83-5d3c98db04a5","added_by":"auto","created_at":"2025-04-22 07:18:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1432743,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6292403/v1/77dfb2e6-5183-4023-9460-bee6eb6f2ab2.pdf"}],"financialInterests":"","formattedTitle":"Bumblebees do not prefer consistent floral scents over variable ones","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMost flowering plants depend on pollinators for reproduction (Klein et al., 2007), so their flowers act like billboards, using various colours, shapes, and scents to attract them (Dobson, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Floral scents, especially, play a key role in pollination (Burkle et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e): scent bouquets attract bees from far away (Raguso, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and often serve as bees\u0026rsquo; primary cue for deciding whether to land on a flower (Kunze et al., 2001; Raguso, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2008a\u003c/span\u003e; Sprayberry, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Yet, despite their influence on bees\u0026rsquo; foraging decisions (Farr\u0026eacute;-Armengol et al., 2015; Larue et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the role of scent in flower choices remains understudied in comparison to flower colours and shapes (Fenster et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Raguso, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2008b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFloral scents consist of complex blends of volatile organic compounds (Knudsen et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), released from various parts of the flower (Pichersky et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Raguso and Pichersky, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). These scent bouquets act as unique floral identifiers (Raguso, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2008a\u003c/span\u003e) and are highly diverse across plant species (Raguso and Pichersky, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Levin et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Scent composition can also vary across plant populations and even among individual flowers within the same species (Raguso et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Burdon et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Delle-Vedove et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the causes of intraspecific scent variation remain poorly studied and understood (Majetic et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Raguso, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), as well as its effects on pollinator foraging behaviour.\u003c/p\u003e \u003cp\u003eScent cues allow flower-visiting insects to associate flowers with pollen and nectar rewards (Kevan and Baker, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Wells and Wells, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Giurfa, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and help them distinguish between plant species (Dobson, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Both honeybees and bumblebees can detect differences in floral scent blends (Laloi and Pham-Del\u0026egrave;gue, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Wright et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and small variations in compound ratios were shown to strongly affect their flower choices (Dobson, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Raguso, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Tan and Nishida, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sol\u0026iacute;s-Montero et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Like floral colours, bees have innate preferences for certain floral volatiles or scent blends (Raguso, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2008a\u003c/span\u003e; Schiestl and D\u0026ouml;tterl, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, it is unclear whether bees also prefer certain patterns of scent variability.\u003c/p\u003e \u003cp\u003eFloral scent is a highly variable trait that plants can adjust to reflect their current state (Dudareva et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Dobson, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). For instance, scented nectar can inform pollinators about reward availability (Heinrich, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Raguso, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Gervasi and Schiestl, 2017). Plants can also modify their scents to better attract pollinators (Dudareva et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Raguso, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2008a\u003c/span\u003e; Leonard et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For example, some flowers adjust the intensity of their scent emissions throughout the day to match pollinator activity (Loughrin et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Raguso, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2008b\u003c/span\u003e; Wright and Thomson, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Scent composition may also vary at different stages of flower development (Majetic et al., 2015; Burkle et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Do bees favour plant species with more consistent or variable scent profiles across flowers?\u003c/p\u003e \u003cp\u003ePollinators not only respond to floral traits but also influence how these traits evolve through selection pressure, including scent (Parachnowitsch et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Schiestl and Johnson, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ollerton et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Stabilising selection driven by pollinator preferences is well-established for flower colour (Goulson, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Whibley et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and similar selection may also shape scent composition (Huber et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mant et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Salzmann et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The pollination syndrome hypothesis suggests that unrelated plant species visited by similar pollinators tend to develop similar floral traits, such as scent composition (Fenster et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Dobson, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Farr\u0026eacute;-Armengol et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), although this idea remains debated (Rosas-Guerrero et al., 2014; Ollerton et al., 2015).\u003c/p\u003e \u003cp\u003eBumblebees tend to specialise and forage on a few flower species (Heinrich, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Chittka et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Such flower constancy benefits plants by ensuring that bees repeatedly visit the same species, increasing pollination efficiency (Heinrich, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Waser, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Like colours, scents promote flower constancy in pollinators (Gegear and Laverty, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gegear, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In turn, this flower constancy probably contributes to stabilising floral traits, including scent profiles (Chittka et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Goulson, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). This tendency to be flower constant may reflect a broader preference for consistency in bee foraging decisions. In fact, bees typically exhibit an aversion to variability and unpredictability in rewards (see review by Anselme, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similarly, we propose that bees may favour plant species with more consistent scent profiles across flowers, potentially placing selection pressure on plants to tightly control their scent composition.\u003c/p\u003e \u003cp\u003eHere, we investigated whether bumblebees (\u003cem\u003eBombus terrestris\u003c/em\u003e) prefer flowers with either consistent or variable scents. To test this, we trained individual bees on two equally rewarding arrays of artificial flowers: all yellow or all green. In one array, all flowers contained sucrose solution scented with a fixed ratio of two artificial food flavourings, creating a consistent scent bouquet. In the other array, flowers had varying ratios of a different pair of scents, making varying, unpredictable scent bouquets. We hypothesized that bees would favour the consistent scent flower, being more predictable than the other flower. We predicted that in a binary choice test, bees would first visit the flower colour associated with the consistent scent.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eColony setup\u003c/h2\u003e \u003cp\u003eCommercial \u003cem\u003eBombus terrestris\u003c/em\u003e colonies were purchased from Koppert (The Netherlands) and kept under controlled laboratory conditions at 22\u0026ndash;24\u0026deg;C with a 14:10 light:dark cycle. Colonies were housed in wooden nestboxes, each connected to its respective flight arena (60 \u0026times; 50 \u0026times; 35 cm) leading to a small chamber (6 \u0026times; 5 \u0026times; 3 cm). The chamber featured a second tube that provided direct access to the arena and was fitted with transparent, removable shutters to regulate bee movement between the nest and arena (see apparatus Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBees were provided daily with pollen balls made from a mix of organic flower pollen pellets and 35% (w/w) sucrose solution, placed directly in the nestboxes. During the day, workers foraged freely on artificial flowers in the flight arena, which offered 35% (w/w) sucrose solution and were regularly refilled. Active foragers were captured and marked on the thorax with uniquely numbered, coloured tags, and considered for selection in the experiment on the same day. A total of 48 bees participated in the experiment from five colonies in May\u0026ndash;June 2022 (see colony details in Supplement \u003cb\u003eS1\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eArtificial flowers\u003c/h3\u003e\n\u003cp\u003eEach artificial flower consisted of a transparent plastic cup with a white lid (height: 4.5 cm, diameter: 3.8 cm), topped with a 2 mm-thick disc of coloured rubber foam. At the centre, a small opaque white resin cup (diameter: 4 mm, depth: 6 mm) was inserted into a pre-cut hole and filled with sucrose solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the experiment, flowers were either yellow (peak reflectance: ~520\u0026ndash;700 nm) or green (peak reflectance: ~530 nm; see \u003cb\u003eSupplement S2\u003c/b\u003e for reflectance curves). We selected these colours for their perceptual similarity while still allowing bees to differentiate them (see control experiment below), and to avoid strong innate preferences typically observed for blue or violet flowers (Gumbert, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Raine and Chittka, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Between experimental sessions, bees were pre-trained on bicoloured, half green half yellow flowers, to ensure they associated sucrose rewards equally with both flower colors used in the experiment (Raine and Chittka, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring training, bees foraged on an array of flowers consisting of a 3 \u0026times; 3 grid of either all-yellow or all-green flowers. In the binary choice test, the array contained 5 green and 5 yellow flowers, arranged in two front rows of three flowers and a third row of four flowers (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e below). Flowers were spaced 3.5 cm apart and mounted on a grey-painted plate matching the all-grey arena. The plate ensured consistent flower placement and allowed for easy flower replacement between foraging bouts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eScented sucrose solutions\u003c/h3\u003e\n\u003cp\u003eArtificial flowers provided 35% w/w sucrose solution at a fixed volume, determined individually for each bee based on an estimate of its honey crop size (see below). Sucrose solutions were scented using strawberry, rose, lemon, or vanilla food flavourings (Seeger, Springe, Germany). We created six artificial scent bouquets by mixing the flavourings in different combinations: strawberry\u0026ndash;rose, lemon\u0026ndash;vanilla, strawberry\u0026ndash;vanilla, lemon\u0026ndash;rose, strawberry\u0026ndash;lemon, and vanilla\u0026ndash;rose. Each combination was prepared in three different ratios: 1:1 (equal parts of both scents), 1:3 (25% scent A, 75% scent B), or 3:1 (75% scent A, 25% scent B).\u003c/p\u003e \u003cp\u003eBees were familiarised with each scent (strawberry, rose, lemon and vanilla) 48 hours before the experiment, by placing filter papers soaked with 25 \u0026micro;L of each food flavouring in the four corners of the nestboxes, allowing airborne scents to disperse. This ensured that bees were not reluctant to collect sucrose solution during the experiment due to unfamiliar scents.\u003c/p\u003e\n\u003ch3\u003eEstimation of crop size\u003c/h3\u003e\n\u003cp\u003eTo determine the appropriate volume of sucrose solution used per flower in the experiment, we first estimated the crop capacity of individual bees. Bee foraged on a 3 \u0026times; 3 array of bicoloured flowers (half green, half yellow), with each flower providing 15 \u0026micro;L of a 35% w/w sucrose solution. We recorded the number of flowers collected over two consecutive foraging bouts and estimated the crop size of the bees by averaging the total volume collected across both bouts. This average volume was then divided by nine, and the resulting volume was used as the reward per flower during the experiment. This ensured that the bees could collect all available rewards in each foraging bout.\u003c/p\u003e\n\u003ch3\u003eTraining\u003c/h3\u003e\n\u003cp\u003eAfter its crop size was estimated, each bee was trained for six consecutive foraging bouts on two different 3 \u0026times; 3 flower arrays, alternating between them in each bout. Each flower array was associated with a flower colour (green or yellow) and a scent combination (strawberry\u0026ndash;rose, lemon\u0026ndash;vanilla, strawberry\u0026ndash;vanilla, lemon\u0026ndash;rose, strawberry\u0026ndash;lemon, or vanilla\u0026ndash;rose).\u003c/p\u003e \u003cp\u003eBees experienced a distinct scent combination for each flower colour; for example, if one array had green flowers scented with strawberry\u0026ndash;lemon, the other had yellow flowers scented with the remaining two scents (e.g., rose\u0026ndash;vanilla). We assigned different scent combinations to each flower colour to ensure clear differentiation between flower types and allowed the use of three different scent ratios (1:1, 1:3, and 3:1) in the variable flower type, while maintaining a consistent 1:1 ratio in the consistent flower (see below).\u003c/p\u003e \u003cp\u003eThe flower arrays were one of two types:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eConsistent flower array\u003c/b\u003e: All flowers in the array had the same scent combination at a fixed 1:1 ratio (equal parts of both scents);\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eVariable flower array\u003c/b\u003e: Flowers in the array had varying scent ratios: some 1:1, some 1:3 (25% scent A, 75% scent B), and some 3:1 (75% scent A, 25% scent B).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eFor example, if a bee experienced green flowers as the consistent array, all green flowers would have strawberry\u0026ndash;lemon in a fixed 1:1 ratio, and the yellow flowers in the variable array would contain rose\u0026ndash;vanilla with varying scent ratios (1:1, 1:3, and 3:1).\u003c/p\u003e \u003cp\u003eHalf of the bees experienced the consistent flower array as green and the variable array as yellow (n\u0026thinsp;=\u0026thinsp;24 bees), and the other half the reverse (n\u0026thinsp;=\u0026thinsp;24 bees). Bees were required to consistently collect sucrose solution from both array types to ensure they perceived each scent combination as an acceptable reward. Flowers were replaced with clean ones each new bout to prevent scent marks from influencing subsequent foraging (Goulson et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Saleh et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBinary choice test\u003c/h2\u003e \u003cp\u003eAfter the six training bouts, each bee was presented with an array of 10 flowers \u0026mdash; 5 yellow and 5 green, arranged so that neighbouring flowers alternated in colour. The flowers were unrewarded and filled with unscented plain water. Bees were randomly assigned one of two array layouts, where the front row contained either two green flowers and one yellow (\u0026ldquo;green-biased\u0026rdquo; layout), or the reverse (\u0026ldquo;yellow-biased\u0026rdquo; layout). We recorded the first flower choices as an indicator of preference.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eControl experiment\u003c/h3\u003e\n\u003cp\u003eA pilot was conducted before the main experiment to test whether bees could differentiate between green and yellow flowers. The setup mirrored the main experiment, where individual bees completed six training bouts to a 3 \u0026times; 3 flower array of a single colour and alternating between arrays each bout, followed by a binary choice test with both flower colours.\u003c/p\u003e \u003cp\u003eInstead of being scented, one flower type offered 25 \u0026micro;L of a low-quality sucrose solution (15% w/w), while the other provided 35% w/w sucrose solution. This clear difference in reward quality allowed us to assess whether bees could correctly identify the flower colour associated with the higher-quality reward. All tested bees (n\u0026thinsp;=\u0026thinsp;10) successfully chose first the flower colour associated with high-quality reward in the final test.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe binary choice test was video-recorded for each bee with a camera (Sony HDR-CX220) positioned above the flight arena. Bee behaviour was analysed using the event-logging software BORIS (v8.6). We tested the hypothesis that bees would favour the flower colour (green or yellow) associated with a consistent scent. To assess this, we recorded (1) the first flower visit and (2) the first 10 flower visits during the binary choice test, as indicators of preference.\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eFor the first flower visit, we built a generalized linear mixed model (GLMM) with a binomial distribution, using first flower scent choice (1 for consistent, 0 for variable) as the response variable and flower colour (green or yellow) as the predictor. Random effects included bee colony, first flower scent encountered (i.e., whether the first training bout involved the consistent or variable array), first flower colour encountered (green or yellow in the first training bout), and binary test layout (\u0026ldquo;green-biased\u0026rdquo; or \u0026ldquo;yellow-biased\u0026rdquo;). Predicted probabilities of first choice were assessed through post hoc pairwise comparisons with a Tukey correction.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eFor the first ten flower visits, we fitted a GLMM with a binomial distribution, using flower scent choices (1 for consistent, 0 for variable) as the response variable and flower colour (green or yellow) as the predictor. Random effects included individual bee identities nested within their colony, first flower scent encountered, first flower colour encountered and binary test layout. Predicted probabilities of flower choices were evaluated through post hoc pairwise comparisons with a Tukey correction.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eData processing was conducted in Python (v3.11, Python Software Foundation, 2023) using the \u003cem\u003epandas\u003c/em\u003e library (McKinney, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) for data structuring and \u003cem\u003eseaborn\u003c/em\u003e (Waskom, 2021) and \u003cem\u003eMatplotlib\u003c/em\u003e (Hunter, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) for data visualization. Statistical analyses were performed in R (v4.1, R Core Team, 2022) using the \u003cem\u003eglmmTMB\u003c/em\u003e package (Brooks et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) for GLMMs, and \u003cem\u003eemmeans\u003c/em\u003e (Lenth, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) for post hoc tests. Model residuals were evaluated with the \u003cem\u003eDHARMa\u003c/em\u003e package (Hartig, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Complete statistical analyses and datasets are available on Zenodo (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5281/zenodo.14993082\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.14993082\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFirst flower choice\u003c/h2\u003e \u003cp\u003e31 of the 48 tested bees (64.6% \u0026plusmn; 8.6%) first visited a yellow flower in the binary choice test, while 17 bees (35.4% \u0026plusmn; 11.6%) chose a green flower. This difference was borderline significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.059, two-tailed exact binomial test, 95% CI: 49.5\u0026ndash;77.8%). Regarding flower scent choice, 27 bees (56.3% \u0026plusmn; 9.5%) chose a flower associated with variable scents, and 21 bees (43.7% \u0026plusmn; 10.8%) a flower associated with a consistent scent.\u003c/p\u003e \u003cp\u003eWe then tested the combined effects of flower scent, colour, and random effects on bees\u0026rsquo; first flower choice (see model (1) in Data analysis). Bees showed no preference between flowers associated with consistent or variable scents (GLMM, binomial family; intercept: Estimate = -0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50, z = -0.72, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.47; N\u0026thinsp;=\u0026thinsp;48; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and flower colour had no significant effect on this choice (X\u0026sup2; = 0.073, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.79).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFirst 10 flower visits\u003c/h2\u003e \u003cp\u003eDuring their first ten visits in the binary choice test, bees visited yellow flowers 51.2% (\u0026plusmn;\u0026thinsp;3.3%) of the time and green flowers 48.8% (\u0026plusmn;\u0026thinsp;3.4%). Regarding flower scent choice, 50.3% (\u0026plusmn;\u0026thinsp;3.3%) of visits were to flowers associated with variable scents, and 49.7% (\u0026plusmn;\u0026thinsp;3.3%) to flowers associated with a consistent scent.\u003c/p\u003e \u003cp\u003eThe GLMM (see model (2) in Data Analysis) revealed no preference between flowers associated with consistent or variable scents (binomial family; intercept: Estimate = -0.007\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, z = -0.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.96; N\u0026thinsp;=\u0026thinsp;457; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and flower colour had no effect on first 10 visits (χ\u0026sup2; = 0.004, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.95).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eScents preferences\u003c/h2\u003e \u003cp\u003eOverall, bees showed no preference for any scent bouquet in the binary choice test (Chi-square goodness-of-fit test: X\u0026sup2; = 1.75, df\u0026thinsp;=\u0026thinsp;5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.88; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and did not favour any individual scent\u0026mdash;lemon, rose, vanilla, or strawberry (X\u0026sup2; = 1.08, df\u0026thinsp;=\u0026thinsp;3, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.78; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe tested whether bees had a preference between two equally rewarding flower arrays, yellow or green, one with consistent scents across flowers and the other with variables scents between flowers. Contrary to expectations, bees showed no preference for flowers with a consistent scent. During training, they readily collected sucrose solution flavoured with varying scent combinations and ratios, and in the binary choice test, they did not favour the flower colour associated with a consistent scent. To our knowledge, this is the first study to explicitly test whether bees prefer consistent or variable scents\u0026mdash;or any other neutral cue like colour or shape\u0026mdash;independently of reward variation.\u003c/p\u003e \u003cp\u003eEvery tested bee collected all sucrose rewards in both flower arrays, regardless of scent composition. This reflects bees' ability to rapidly associate scents with rewards (Kunze and Gumbert, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Wright and Schiestl, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Giurfa, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), even when they have innate preferences for specific scent volatiles (Milet-Pinheiro et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Raguso, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2008a\u003c/span\u003e). Surprisingly, bees visited and accepted every flower in the variable array, despite previous findings that bumblebees exhibit even stronger flower constancy when flowers differ in multiple sensory cues, such as scents and colours (Wells and Wells, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Gegear and Laverty, 2001; Gegear, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, recent research has challenged the idea of high flower constancy in bumblebees; Yourstone et al. (\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) found that bees were less flower constant than expected, with only 23% of their foraging trips occurring on the same flower species.\u003c/p\u003e \u003cp\u003eIn the binary choice test, bees showed a slight tendency to visit yellow flowers first, though this preference was not significant. Bees are known to have an innate preference for yellow flowers (Lunau, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Gumbert, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). To ensure that choices were based on learned associations, we used unscented, unrewarded flowers in the binary test. Hence it is unclear whether scent preferences would have outweighed colour preferences if both cues were present simultaneously. Notably, Larue et al. (2015) found that floral scent had a stronger influence than visual traits in attracting flower-visiting insects.\u003c/p\u003e \u003cp\u003eWere bees in our experiment able to distinguish between scent combinations, particularly the different scent blend ratios in the variable flower array? Studies show that bees can detect individual volatiles within complex scent blends, influencing their flower choices (Laloi and Pham-Del\u0026egrave;gue, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Locatelli et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and honeybees can even perceive subtle differences in the ratio of two scents (Wright et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Bateson et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Moreover, since our flowers contained scented sucrose solution, bees could also taste them; Robertson (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) suggests that olfactory and gustatory receptors may overlap in bumblebees. In nature, scented nectar contains volatile organic compounds, which pollinators use to assess reward availability before landing on a flower (Heinrich, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Raguso, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2008b\u003c/span\u003e), and can impact their foraging behaviour (Raguso, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Given this, it seems likely that bees could sense the variability in the variable flowers, and distinguish this from the fixed-ratio flowers.\u003c/p\u003e \u003cp\u003eAlthough no study has directly tested bees\u0026rsquo; preference for consistency in neutral cues, some have examined their responses to inconsistent rewarding cues. Honeybees tended to avoid choices where uncertain visual cues predict reward or punishment (Perry and Barron, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Andrew et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) found that honeybees trained to discriminate between a rewarding and a punishing scent blend preferred a novel scent that was more distinct from the punishing one, rather than similar to the reward. Likewise, Lynn et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) showed that bumblebees favoured novel colours that minimised the risk of choosing an unrewarding flower. This phenomenon, known as \u0026ldquo;peak shift\u0026rdquo; (Hanson, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1959\u003c/span\u003e), occurs when animals develop a preference for a more extreme version of a rewarded stimulus to avoid similar, non-rewarded ones. These findings suggest that bees may prefer consistent cues over variable ones, to reduce uncertainty. Stress may further amplify this avoidance of inconsistent cues: Bateson et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) found that shaken honeybees were less likely to respond to ambiguous scents associated with rewards.\u003c/p\u003e \u003cp\u003eWhen it comes to rewards preferences, bees are generally risk-averse and tend to avoid variability (see review by Anselme, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). They typically prefer consistent nectar amounts over variable ones (Real, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Waddington et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Shafir et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), which is likely in part due to the psychophysics of reward perception (Kacelnik and Bateson, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). When resources are variable, bumblebees also forage less efficiently (Dunlap et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and rely more on social information for flower choice (Smolla et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, their sensitivity to variability depends on context. In some contexts, bees showed indifference to fluctuations in nectar concentration or volume (Waddington, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e1995\u003c/span\u003el\u0026ouml;p and Menzel, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and when variable distribution did not include null rewards (Drezner-Levy and Shafir, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). They may even favour variable rewards when advantageous: For example, bumblebees initially preferred consistent rewards but shifted to variable ones when colony nectar reserves were low, both in natural foraging conditions (Cartar, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) and in laboratory settings (Cartar and Dill, 1990).\u003c/p\u003e \u003cp\u003eBees' preferences for specific floral volatiles are well-documented (Knudsen et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Farr\u0026eacute;-Armengol et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Benelli et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bisrat and Jung, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and they readily associate these scents with rewards (Wells and Wells, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Wright and Schiestl, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Giurfa, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Bees can even retain scent memories longer than visual cues (Menzel, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Kunze and Gumbert, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Bumblebees, in particular, use floral scents as social cues, with foragers transferring scent compounds within the nest to inform nestmates (Molet et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). They also favour flowers that match the scents collected by successful foragers (Dornhaus and Chittka, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, bees learn consistent scents more effectively than variable ones (Wright and Thomson, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Wright and Schiestl., 2009). For instance, Wright et al. (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) found that honeybees reject scent-modified flowers even if they contain familiar scent compounds, highlighting the importance of scent consistency for pollinator recognition. Many rewardless flowers emit weak or highly variable scent, likely to avoid detection by scent-learning pollinators (Jers\u0026aacute;kov\u0026aacute; and Johnson, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Salzmann et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Salzmann et al. (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) found that rewarding orchids produce strong, consistent scents that bees can detect, whereas deceptive orchids emit weak, highly variable scents. Similarly, floral compounds that attract pollinators tend to be more consistent across populations and species, while non-attractive compounds show greater variability (Mant et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Huber et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNonetheless, floral scent remains a highly variable trait (Dudareva et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), even among individual flowers within the same species (Burdon et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). While plants adjust scent emissions to attract pollinators (Majetic et al., 2015; Burkle et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), scent bouquets are also influenced by environmental factors (Dudareva et al., 1999; Raguso, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2008b\u003c/span\u003e). Studies suggest that intraspecific variation in floral scent may help attract local pollinators (Soler et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Larue et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Vega-Polanco et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). F\u0026uuml;l\u0026ouml;p and Menzel (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) propose that bees\u0026rsquo; tolerance for scent variability may be an adaptation to cope with unreliable floral resources.\u003c/p\u003e \u003cp\u003eFloral scents, including scented nectar, likely serve as honest signals to pollinators (Howell and Alarc\u0026oacute;n, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Gervasi and Schiestl, 2017). Bees often select flowers based on their scent composition (Pichersky and Raguso, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Knudsen and Gershenzon, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) or the intensity of scent emissions (Majetic et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Parachnowitsch et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), which inform foragers about nectar and pollen availability. We suggest that bees are likely to respond to olfactory cues regardless of whether they are more variable or consistent, as long as they can reliably associate them with rewards.\u003c/p\u003e \u003cp\u003eOur findings highlight bees' ability to rapidly learn scent-reward associations and show that they forage equally on flower arrays with both variable and consistent scents, and show no preference between them. This in turn suggests that plants may not be under strong selection to reduce inter-flower scent variability, at least in terms compound ratios. Maintaining tight control over a trait in the face of environmental variation is costly, and if no selection for this maintenance is present, we would expect such traits to fluctuate, as indeed is the case with intraspecific scent variation between flowers (Burdon et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Delle-Vedove et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, our controlled experiment likely oversimplified the complex sensory landscape bees navigate in nature, and further research is needed to understand how they respond to scent variability within flower patches. Despite its importance, the role of floral scent in bee foraging has been largely overlooked (Fenster et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Raguso, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, 2008). More generally, future studies should examine whether bees' aversion to variability in rewards extends to other neutral cues like morphology or colour, as the foraging preferences of plant pollinators play a key role in shaping floral trait evolution (Ollerton et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSUPPLEMENTARY MATERIAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS1\u003c/strong\u003e contains the dataset of the main experiment, \u003cstrong\u003eS2\u003c/strong\u003e provides the reflectance curves of the artificial flowers, and \u003cstrong\u003eS3\u003c/strong\u003e the statistical analysis of the experiment. All supplementary materials are available on Zenodo (https://doi.org/10.5281/zenodo.14993082).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to thank A. Koch and M. Kietniz for their assistance with data collection, and K.\u0026nbsp;Hartmannsgruber for video analysis. Special thanks to A. Avargu\u0026egrave;s-Weber for providing spectrometer measurements of the artificial flowers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. A. was supported by an ERC Starting Grant to T. J. C. [H2020-EU.1.1. #948181] and T. J. C. was supported by a Heisenberg Fellowship from the Deutsche Forschungsgemeinschaft [#462101190].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS\u0026rsquo; CONTRIBUTION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM\u0026eacute;lissa Armand:\u003c/strong\u003e Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Writing - Original Draft, Writing - Review and Editing, Visualization. \u003cstrong\u003eLisa Zeilmann:\u003c/strong\u003e Investigation. \u003cstrong\u003eChristian Weinzettl\u003c/strong\u003e: Investigation. \u003cstrong\u003eTomer J. Czaczkes:\u003c/strong\u003e Conceptualization, Methodology, Validation, Resources, Writing - Review and Editing, Supervision, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no conflicts of interest in the production of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndrew SC, Perry CJ, Barron AB et al (2014) Peak shift in honey bee olfactory learning. 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Behav Ecol 34:602\u0026ndash;612. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/beheco/arad028\u003c/span\u003e\u003cspan address=\"10.1093/beheco/arad028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6292403/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6292403/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo attract pollinators, flowering plants evolve diverse sensory traits into compelling signals. Floral scent, in particular, plays a key role in drawing bees from a distance and shaping their foraging choices. Scent composition varies widely, including across flowers of the same plant species. Yet, it is unclear whether scent variability influences bee flower choices, and thus whether plants would be under selection to minimise variation in their scent composition. Since bees typically avoid variability in rewards, we hypothesised they would favour flowers with more consistent scents. To test this, we trained individual bumblebees (\u003cem\u003eBombus terrestris\u003c/em\u003e) on two equally rewarding flower arrays: one with a consistent scent blend across flowers, and the other with variable scent blends between flowers. Contrary to expectations, bees showed no preference for scent consistency. They readily foraged from both arrays across bouts and did not favour either flower type in the binary choice test. To the best of our knowledge, this is the first study to examine how bees respond to scent variability. A better understanding of scent profile preferences in pollinators could offer new insights into their co-evolution with plants and the development of floral traits. More broadly, further research is needed on how pollinators respond to variability and unpredictability in neutral cues like scent or colour, a largely overlooked aspect of foraging decision-making.\u003c/p\u003e","manuscriptTitle":"Bumblebees do not prefer consistent floral scents over variable ones","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 07:02:25","doi":"10.21203/rs.3.rs-6292403/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-04-22T23:30:16+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-03T16:40:55+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T13:27:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-01T12:48:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Insectes Sociaux","date":"2025-03-29T03:29:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"insectes-sociaux","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inso","sideBox":"Learn more about [Insectes Sociaux](http://link.springer.com/journal/40)","snPcode":"40","submissionUrl":"https://www.editorialmanager.com/inso/default2.aspx","title":"Insectes Sociaux","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cdd78ac5-2e08-44b9-861c-95e058930912","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T01:35:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-22 07:02:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6292403","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6292403","identity":"rs-6292403","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}