Evaluation of electrophysiological responses of bumblebee pollinators to floral scents of oil pumpkin

Evaluation of electrophysiological responses of bumblebee pollinators to floral scents of oil pumpkin | Research Square 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of electrophysiological responses of bumblebee pollinators to floral scents of oil pumpkin Monica Barman, Raimund Tenhaken, Stefan Dötterl This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8764185/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bumblebees are important pollinators of wild and crop plants, among them the Styrian oil pumpkin ( Cucurbita pepo subsp. pepo var. styriaca Greb.). The flowers of this economically important crop species release strong scents, dominated by 1,4-dimethoxybenzene, which are believed to be involved in the attraction of the bumblebees to the flowers. To determine the volatiles that elicit physiological responses in the antennae of Bombus terrestris , the most important bumblebee pollinator species of Styrian oil pumpkin in Europe, we performed gas chromatography-electroantennographic detection (GC-EAD) analyses using both natural floral scent samples and synthetic blends. The synthetic blend contained a mixture of 21 volatile compounds in similar amounts, independent of whether they have been dominant or minor compounds in the natural samples. From the measurements with natural samples, we found that only 1,4-dimethoxybenzene elicited strong responses, with few other compounds eliciting only weak responses. However, in the measurements with the synthetic blends, most of the 21 compounds including terpenoids and benzenoids/phenylpropanoids, showed similar responses as 1,4-dimethoxybenzene. This study demonstrates that the antennae of bumblebees respond to a high number of floral scent compounds of pumpkin, which all have the potential to attract pollinators and thus contribute to fruit set of this commercially important crop. The compounds identified as being EAD-active in the present study can be used in future studies to determine the effects of these compounds, as a mixture and as individual compounds, in attracting bumblebee pollinators to pumpkin flowers. Pumpkins floral scents bumblebees gas chromatography-electroantennography (GC-EAD) antennal response Figures Figure 1 Figure 2 Introduction Insect pollinators are attracted to flowers via visual (e.g., color) as well as olfactory (scent) cues (Borghi et al. 2017 ), and play a key role in ensuring fruit set not only in most of the wild flowering plants, but also in many economically important crops (Klein et al. 2007 ; Ollerton et al. 2011 ). Examples for the latter are commercial cucurbit species that bear unisexual flowers and rely on insect pollinators, including bumblebees (e.g., Bombus terrestris in Europe), for pollen transfer from male to female flowers (Barman et al. 2023 ; Bomfim et al. 2016 ). The present study focuses on Styrian oil pumpkin ( Cucurbita pepo subsp. pepo var. styriaca Greb.), which is a monoecious plant species bearing unisexual male and female flowers on a plant individual. For over 100 years, this variety is widely cultivated in Styria, a south-eastern region in Central Europe, and meanwhile also across various parts of Europe and Asia, for obtaining premium-quality seed oil (Fruhwirth & Hermetter 2007 ). In Central Europe, main pollinators of this pumpkin variety are honeybees, while bumblebees have been found to be a suitable alternative in cases where honeybee or other wild bee populations are available to a limited extent only (Fuchs & Müller 2004 ). Our recent findings on the floral scent of Styrian oil pumpkin (Barman et al. 2024 ) and studies on other commercial pumpkin cultivars (e.g., Andrews et al. ( 2007 ) revealed that both male and female pumpkin flowers are strongly scented, typically having one or few strongly dominant compounds (e.g., 1,4-dimethoxybenzene), but release also various benzenoids/phenylpropanoids (e.g., cinnamaldehyde, benzyl alcohol, p -anisaldehyde, 1,2,4-trimethoxybenzene), terpenoids (e.g., linalool, linalool oxides) and nitrogen-containing compounds (e.g., phenylacetonitrile, 1-nitro-2-phenylethane), most of them in minor or trace amounts. Some compounds thereof, such as 1,2,4-trimethoxybenzene and cinnamaldehyde, have been shown to attract specialist bee pollinators to the cucurbit flowers (Andrews et al. 2007 ). Additionally, based on work with other plant species, that however, released some of the compounds also known from cucurbits, it is known that some of the cucurbit volatiles, such as p -anisaldehyde and 1,4-dimethoxybenzene, elicit physiological responses in the antennae of generalist bumblebees (Salzmann et al. 2007 ). However, for most of the volatiles released from pumpkin flowers, it is neither known whether specialist or generalist cucurbit pollinators can detect them, nor has it been studied which of the volatiles are involved in attracting generalist pollinators. This study, for the first time, determined antennal responses of a cucurbit pollinator, i.e., generalist B. terrestris , to the flower scents of a high-value oil pumpkin species. Specifically, using gas chromatography coupled with electroantennographic detection (GC-EAD), we tested antennae of B. terrestris on a natural flower scent sample of Styrian oil pumpkin and on synthetic mixtures containing similar amounts of compounds, that had been identified as flower scent of this species in our previous study (Barman et al. 2024 ). This knowledge base is an important step in understanding the communication between this pollinator-dependent crop and a main pollinator, which might be used in the future to manipulate the floral scent in order to improve its fruit set. Materials and Methods Plant material and bumblebees Styrian oil pumpkin ( Cucurbita pepo subsp. pepo var. styriaca Greb.) seeds were obtained from Saatzucht Gleisdorf GmbH, Austria and plants were grown and maintained in the greenhouse facility of the Botanical Garden, University of Salzburg, throughout the summer months of 2023. For electroantennographic experiments, a Bombus terrestris bumblebee colony was obtained from Biobest® Belgium NV and kept in a flight cage (4 m width × 8 m length × 2.20 m height; Rachersberger et al. 2019 ) built in the Botanical Garden of the University of Salzburg. Natural scent samples To obtain natural scent samples for GC-EAD (Gas chromatography coupled to electroantennographic detection) studies, scents were collected from flowers of Styrian oil pumpkin plants ( n = 5 ; from five different plant individuals). We utilized male pumpkin flowers for scent collection, mainly due to higher availability of this flower sex, and also because both male and female flowers did not show difference in the scent profile (Barman et al. 2024 ). Following their opening at 6:00 in the morning, a total of 4–5 flowers were together placed in a clean oven bag (Toppits®, Minden, Germany; dimensions: 30 cm × 30 cm). The scent was collected for 4 hours in adsorbent glass tubes (length: 8 cm, inner diameter: 2.5 mm), containing 30 mg mixture (1:1) of Tenax TA and Carbotrap B (both from Supelco, Merck KGaA, Darmstadt, Germany), with the help of a rotary vane pump (G12/01 EB; Rietschle Thomas Inc., Puchheim, Germany; flow of 200ml per min). The volatiles trapped were eluted with 80 µL of high-grade acetone (Rotisolv, Roth, Germany) and stored at − 20°C until the GC-EAD measurements. Of the five natural scent samples collected, the one sample that contained the highest concentration of scents was selected for the GC-EAD analyses. Synthetic scent samples Based on the floral scent data of Styrian oil pumpkin ( Cucurbita pepo subsp. pepo var. styriaca Greb.) of a previous study (Barman et al. 2024 ), we prepared three blends (to avoid overlapping of compounds in the GC runs) of overall 21 synthetic chemicals (including a few stereoisomers), all diluted 1000-fold (v/v) in pure grade acetone (the same as above) to achieve the same concentration, and obtained from Sigma-Aldrich Chemie GmbH, with greater than or equal to 97% purity. Blend 1 contained p -methylanisole, ( S )-linalool, ( R )-linalool, benzyl alcohol, phenylacetonitrile, p -anisaldehyde, 3,4-dimethoxystyrene, p -anisyl alcohol, ( E )-cinnamyl alcohol, ( E )- p -methoxycinnamaldehyde, benzyl benzoate, and 4-hydroxy-3-methoxy-cinnamaldehyde. Blend 2 contained p -vinylanisole, linalool oxide (isomer mix), 2-phenylethanol and ( E )-cinnamaldehyde, and blend 3 contained 1,4-dimethoxybenzene, epoxyoxoisophorone, 4-oxoisophorone, benzenepropanol, and 1,2,4-trimethoxybenzene. Electrophysiological assays The electroantennographic measurements were performed by the method as described in Heiduk et al. ( 2016 ). Physiologically active compounds were identified by injecting 1 µL of the natural headspace floral scent sample or the synthetic blends in a gas chromatograph (Agilent 7890 A Santa Clara, CA, USA) equipped with a flame ionization detector (FID). For the analysis, a chiral MEGA-DEX DMT Beta SE column (30m × 0.25mm ID, 0.23µm film thickness) was used. The column is split at the end by a µFlow splitter into two capillaries of which one leads to the FID (2 m x 0.15 µm inner diameter) and the other one to the EAD (1 m x 0.2 µm inner diameter) setup. For preparing an antenna for measurements, we utilized the protocol as mentioned in Dötterl et al. ( 2005 ). Briefly, an antenna was excised from the bumblebee worker and placed within glass micropipette electrodes filled with insect ringer (8.0 g/l NaCl, 0.4 g/l KCl, 0.4 g/l CaCl 2 ). These electrodes were connected to silver wires. A total of 15 EAD runs were performed with the antennae of five B. terrestris individuals with natural scent samples. For the GC-EAD runs with synthetic blends, 5 EAD runs were recorded for blend 1 using antennae from 2 bee individuals, blends 2 and 3 had 3–4 runs each, also using antennae from two bees per blend. A compound was treated as being EAD-active when it elicited a response in at least half of the runs, where this specific compound has been tested. Results and Discussion In the GC-EAD measurements with the natural headspace floral scent sample, the benzenoid 1,4-dimethoxybenzene (1,4-DMB), which, as expected (Barman et al. 2024 ), strongly dominated the sample, elicited the strongest response in the antennae of Bombus terrestris. Weak responses were elicited by ( R )-linalool, p -vinylanisole and 4-oxoisophorone present in natural sample (Fig. 1 ). Compounds such as benzyl alcohol, phenylacetonitrile, benzenepropanol, 1,2,4-trimethoxybenzene and 1- nitro-2-phenylethane that were also present as minor/trace compounds in the sample, did not elicit antennal responses (Fig. 1 ), though some of them (e.g., benzyl alcohol, phenylacetonitrile) are known to elicit physiological and / or behavioural responses in bees (e.g., Rachersberger et al. 2019 ; Jürgens et al. 2014 ; Burger et al 2021 ; Dötterl and Gershenzon 2023). The compound 1,4-DMB does not only occur in Styrian oil pumpkin, but is the most commonly emitted compound among several other commercially cultivated cucurbits (reviewed in Barman et al. 2023 ) as well as in some non-cucurbit flowers, and our finding that this benzenoid is physiologically active in antennae of bumblebees is consistent with previous findings indicating that this compound is an attractant for bumblebees and for other generalist as well as for specialist bees (Dötterl et al. 2005 ; Salzmann et al. 2007 ; Jürgens et al. 2014 ; Perkins et al. 2023 ). In the measurements with the synthetic blends, that overall contained 21 compounds known from Styrian oil pumpkin (Barman et al. 2024 ) in equal concentrations, most of the compounds, elicited comparable strong responses as 1,4-dimethoxybenzene in antenna of B. terrestris (Fig. 2 ). Among them the benzenoids/phenylpropanoids p -anisaldehyde, p -anisyl alcohol, benzenepropanol, benzyl alcohol, benzyl benzoate, ( E )-cinnamaldehyde, ( E )-cinnamyl alcohol, and 3,4-dimethoxystyrene, and the terpenoids linalool (both S and R stereo-isomers, although typically ( R )-linalool is found in natural sample) and epoxyoxoisophorone. Some of compounds, especially ( E )- p -methoxycinnamaldehyde, elicited only weak responses. Weak responses were also elicited by different isomers of linalool oxide and 4-hydroxy-3-methoxy-cinnamaldehyde, which were available in smaller amounts in the scent samples than the other compounds (Fig. 2 ). Out of all the EAD-active compounds, only 1,4-DMB, p -anisaldehyde and linalool have been previously reported to elicit responses in the antenna of bumblebees (Salzmann et al. 2007 ; Dötterl and Gershenzon 2023), but a few other compounds, such as benzyl alcohol and ( E )-cinnamyl alcohol, are known to be physiologically active in antennae of the related Western honeybee, Apis mellifera (Rachersberger et al. 2019 ). The compounds 1,2,4-trimethoxybenzene and ( E )-cinnamaldehyde are attractive to the specialist squash bees ( Peponapis pruinosa ), making it possible that they are also behaviourally active in bumblebees. Among the EAD-active compounds are also compounds such as epoxyoxoisophorone that is an effective attractant for butterflies (Lehner et al. 2022 ), and behavioural assays with bumblebees are needed to test whether these compounds are attractive to bumblebees. Conclusions Our experiments revealed that many of compounds released by flowers of Styrian oil pumpkin are physiologically active in the antennae of one of its important pollinators in Central Europe, the bumblebee Bombus terrestris , and potentially involved in attracting this bee species to the flowers. With very few exceptions, most of the compounds were not known to being physiologically active in antennae of bumblebees before this study. Measurements with a natural flower scent sample that contained only a single main compound and most of the scent compounds in minor/trace amounts, revealed only a few of the compounds as being EAD-active. Their amounts were obviously too small to elicit responses above detection threshold, given that they elicited strong responses in our measurements with synthetic samples that contained the compounds in similar (high) amounts. Thus, compounds potentially involved in the communication between plants and their (bee) pollinators might be overlooked when performing EAD measurements only with natural scent samples, especially if these samples contain compounds in trace amounts only. However, behavioural experiments are still needed to test which of the many EAD-active compounds are involved in attracting B. terrestris and potentially other bee pollinators to oil pumpkin flowers. Such compounds might be applied to pumpkin fields to enhance pollinator visitation and fruit set, as fruit and seed set in Styrian oil pumpkin is largely pollinator limited (Fuchs and Müller 2004 ). Declarations Conflicts of interest The authors hereby declare that they have no conflict of interest. Funding This research work was funded in whole or in part by the Austrian Science Fund (FWF) (doi: 10.55776/M3233 ). Author Contribution Monica Barman: Conceptualization, Methodology, Investigation, Visualization, Funding acquisition, Project administration, Writing – original draft, Writing – review & editing. Raimund Tenhaken: Funding acquisition, Validation, Writing – review & editing. Stefan Dötterl: Conceptualization, Validation, Funding acquisition, Project administration, Writing – review & editing. Acknowledgement We thank the gardeners at the Botanical Garden of Salzburg University for their assistance in maintaining the experimental plants and Roman Fuchs for his technical assistance during GC-EAD analysis. References Andrews ES, Theis N, Adler LS (2007) Pollinator and herbivore attraction to Cucurbita floral volatiles. J Chem Ecol 33(9):1682–1691. https://doi.org/10.1007/s10886-007-9337-7 Barman M, Tenhaken R, Dötterl S (2023) A review on floral scents and pigments in cucurbits: Their biosynthesis and role in flower visitor interactions. Sci Hortic 322:112402. https://doi.org/10.1016/j.scienta.2023.112402 Barman M, Tenhaken R, Dötterl S (2024) Negative and sex-specific effects of drought on flower production, resources and pollinator visitation, but not on floral scent in monoecious Cucurbita pepo . New Phytol 244(3):1013–1023. https://doi.org/10.1111/nph.20016 Bomfim I, Freitas B, Aragão F, Walters A (2016) Pollination in cucurbit crops. In: Pessarakli M (ed) Handbook of Cucurbits: Growth, Cultural Practices, and Physiology. CRC, pp 181–200. https://doi.org/10.1201/b19233-16 Borghi M, Fernie AR, Schiestl FP, Bouwmeester HJ (2017) The sexual advantage of looking, smelling, and tasting good: the metabolic network that produces signals for pollinators. Trends Plant Sci 22(4):338–350. https://doi.org/10.1016/j.tplants.2016.12.009 Burger H, Joos N, Ayasse M (2021) Floral cues of non-host plants attract oligolectic Chelostoma rapunculi bees. Front. Ecol Evol 9:682960. https://doi.org/10.3389/fevo.2021.682960 Dötterl S, Füssel U, Jürgens A, Aas G (2005) 1,4-Dimethoxybenzene, a floral scent compound in willows that attracts an oligolectic bee. J Chem Ecol 31(12):2993–2998. https://doi.org/10.1007/s10886-005-9152-y Dötterl S, Jürgens A, Seifert K, Laube T, Weißbecker B, Schütz S (2006) Nursery pollination by a moth in Silene latifolia : the role of odours in eliciting antennal and behavioural responses. New Phytol 169:707–718. https://doi.org/10.1111/j.1469-8137.2005.01509.x Fruhwirth GO, Hermetter A (2007) Seeds and oil of the Styrian oil pumpkin: Components and biological activities. Eur J Lipid Sci Technol 109(11):1128–1140. https://doi.org/10.1002/ejlt.200700105 Fuchs R, Müller M (2004) Pollination problems in Styrian oil pumpkin plants: Can bumblebees be an alternative to honeybees? Phyton 44(1):155–165 Heiduk A, Brake I, von Tschirnhaus M, Göhl M, Jürgens A, Johnson SD, Meve U, Dötterl S (2016) Ceropegia sandersonii mimics attacked honeybees to attract kleptoparasitic flies for pollination. Curr Biol 26(20):2787–2793. https://doi.org/10.1016/j.cub.2016.07.085 Jürgens A, Glück U, Aas G, Dötterl S (2014) Diel fragrance pattern correlates with olfactory preferences of diurnal and nocturnal flower visitors in Salix caprea (Salicaceae). Bot J Linn Soc 175(4):624–640. https://doi.org/10.1111/boj.12183 Klein AM, Vaissière BE, Cane JH, Steffan-Dewenter I, Cunningham SA, Kremen C, Tscharntke T (2007) Importance of pollinators in changing landscapes for world crops. Proc. R. So. B. Biol Sci. 274(1608):303–313. https://doi.org/10.1098/rspb.2006.3721 Lehner S, Schulz S, Dötterl S (2022) The mystery of the butterfly bush Buddleja davidii : How are the butterflies attracted? Front Plant Sci 13:994851. https://doi.org/10.3389/fpls.2022.994851 McFrederick QS, Kathilankal JC, Fuentes JD (2008) Air pollution modifies floral scent trails. Atmos Environ 42(10):2336–2348. https://doi.org/10.1016/j.atmosenv.2007.12.033 Ollerton J, Winfree R, Tarrant S (2011) How many flowering plants are pollinated by animals? Oikos 120(3):321–326. https://doi.org/10.1111/j.1600-0706.2010.18644.x Perkins J, Hayashi T, Peakall R, Flematti GR, Bohman B (2023) The volatile chemistry of orchid pollination. Nat Prod Rep 40(4):819–839. https://doi.org/10.1039/D2NP00060A Rachersberger M, Cordeiro GD, Schäffler I, Dötterl S (2019) Honeybee pollinators use visual and floral scent cues to find apple ( Malus domestica ) flowers. J Agric Food Chem 67(48):13221–13227. https://doi.org/10.1021/acs.jafc.9b06446 Salzmann CC, Nardella Am Fau - Cozzolino S, Cozzolino S, Fau - Schiestl FP, Schiestl FP (2007) Variability in floral scent in rewarding and deceptive orchids: the signature of pollinator-imposed selection? Ann Bot 100(Print):0305–7364. 757–765. https://doi.org/10.1093/aob/mcm161 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies 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

Related documents

• Floral colour change in Byrsonima variabilis (Malpighiaceae) as a visual cue for pollen but not oil foraging by oil-collecting bees.#647219
• Identification of two odorant receptors tuned to alarm pheromone compounds in the honey bee Apis mellifera#408723
• Floral Phytochemistry: Impact of Volatile Organic Compounds and Nectar Secondary Metabolites on Pollinator Behavior and Health#137643
• Pollinators in blackcurrant orchards: complementary roles of sown and spontaneous flower strips and limited spillover of small wild bees within fields.#1039061
• A matter of smell: The complex regulation of aroma production in melon#172973