2.1 Crop species and pollinators
Strawberry (Fragaria x ananassa Duch - Rosaceae, variety - Florian F1, Dürr-Samen company) is a fruit crop widely appreciated for its characteristic aroma and sweetness. Adequate bee pollination increases yield and fruit quality (Klatt et al. 2014). We used the main pollinators of strawberry for our experiments: Apis mellifera Linnaeus, 1758 (obtained from a local beekeeper), Bombus terrestris Linnaeus, 1758 (Biobest®, Belgium), and Osmia bicornis Linnaeus, 1758 (Mauerbienenzucht®, Germany) (Klein et al. 2007).
Strawberry is a gender-dimorphic species with hermaphroditic and female flowers, but in contrast to wild strawberry (Ashman et al. 2005), we did not find an effect of flower sex on scent emission (total amount of scent: Z= -0.32, N = 10, p = 0.761; relative scent composition: Pseudo-F1,9=1.12, p = 0.323) and thus did not discriminate between the flower sexes in the present study.
2.2 Crop plant cultivation and temperature regime
The strawberry plants were cultivated in two plant growth chambers (Liebherr, Profi line, Germany; adapted with a multistage temperature controller, model TAR 1700-2, Elreha, Germany, and light timer switch, model D21ASTRO 230 V 50/60 Hz, Legrand, Germany) that differed in their temperature setting (optimum and increased, see below). The seeds were randomly assigned to one of the chambers and sown in pots (9 x 8 x 9 cm) using standard soil (Einheitserde®, Profi Substrat) and fertilized once with 50 ml/pot (Wufax®, Nitrogen: 12%, Phosphate: 4%, Potassium: 6%) when plants reached mid-age.
The requirements of strawberry plants for light conditions and water availability were controlled following literature data (Hytönen and Kurokura 2020). Plants were cultivated in both chambers at 16 h light/08 h darkness, and the light intensity was 2000 lx (via cool white led lamps, model VT-5959 LED-Flutlicht, V-TAC, 50 W). The air humidity ranged between 60–70%. To keep the soil at comparable moisture levels during the development of the plants and between the different treatments, as measured by a tensiometer (model FDA 602 TM2, ALMEMO®, Germany), the water supply varied according to the age of the plants and the temperature scenario. When they were sown, the amount was, independent of the scenario, 15 ml/plant/day, in the mid-ages 60 ml/plant/day (optimum scenario) and 90 ml/plant/day (warmer scenario), and, during the flowering phase, 120 ml/plant/day (optimum scenario) and 170 ml/plant/day (warmer scenario).
The plants were grown under two temperature scenarios: optimum and 5°C higher than optimal temperatures (according to the global warming scenario SSP-8.5, IPCC 2021). The mean optimal temperature for the growth and flowering of cultivated strawberry (F. ananassa) plants is 20°C (Hytönen and Kurokura 2020). Considering the mean daily thermal amplitude in Central Europe (PlavcovÁ and KyselÝ 2011; Worldclim 2020), strawberry plants were cultivated in the optimal scenario at day and night with temperatures of 23°C and 13°C (mean 20°C, when considering the length of the day and night periods), respectively, and in the warmer scenario, the temperatures were 28°C and 18°C (mean 25°C) during day and night, respectively. For each temperature scenario, 12 individual plants were cultivated.
2.3 Sampling and analysis of flower scents
Sampling of scent samples was performed inside the growth chambers by dynamic headspace. From 12 individuals cultivated for each scenario, 10 individuals of the optimum scenario and nine individuals of the warmer scenario produced flowers during the experiment and were sampled. The samples were obtained from flowers at the beginning of their first day of anthesis, throughout the day depending on flower opening. A single flower per sample was enclosed in a polyester oven bag (Toppits®). After bagging, two small adsorbend tubes were inserted into the bag: one was used to trap the floral scent, and the other (glass vial filled with 5 mg Carbotrap B) was used to insert clean air from outside of the growth chambers to avoid internal air contamination). The samplings lasted 30 min using membrane pumps (G12/01 EB; Gardner Denver Thomas GmbH, Fürstenfeldbruck, Germany). This time period was enough to obtain the maximum number of compounds as determined by preliminary analyses that used sampling times between 15 min and 2 h. The flows of both pumps were adjusted at 200 ml/min with the help of flowmeters. The adsorbend tubes (quartz vials, length: 25 mm, inner diameter: 2 mm) were filled with 1.5 mg Tenax-TA (mesh 60–80) and 1.5 mg Carbotrap B (mesh 20–40, both Supelco). The adsorbends were fixed in tubes using glass wool. Dynamic headspace samples of green leaves (N = 3 samples per scenario) were collected with the same method to discriminate between vegetative (not considered for subsequent analyses) and flower-specific scent components. Samples from empty oven bags (N = 3) inside the growth chambers were collected to identify potential contaminants.
Scent samples were analyzed using GC/MS (gas chromatography/mass spectrometry). The system consisted of an automated thermal desorption system (model TD-20, Shimadzu, Japan) coupled to a QP2010 Ultra EI GC/MS (Shimadzu, Japan) equipped with a Zebron™ ZB-5 fused silica column (5% phenyl 95% dimethylpolysiloxane; 60 m long; inner diameter 0.25 mm; film thickness 0.25 µm; Phenomenex), as described previously (Mitchell et al. 2015). The GC/MS data were processed using GCMSsolution (Version 4.41, Shimadzu Corporation 2015). The tentative identification of compounds was carried out using the mass spectral libraries Wiley 9, Nist 2011/FFNSC 2, and Adams (2007), as well as the database available in MassFinder 3. The identity of all compounds was confirmed by a comparison of mass spectra and retention times with those of authentic standard compounds available at the Plant Ecology lab of the Paris-Lodron University of Salzburg. To determine the amount of scent trapped, known amounts of monoterpenes, aliphatics, and aromatics were added to clean adsorbent tubes and analyzed by GC/MS as described above; mean peak areas (total ion current) of these compounds were used to determine the total amount of strawberry scent (Cordeiro et al. 2019).
2.4 Synthetic scent sample
A synthetic mixture with four compounds (benzyl alcohol, methyl salicylate, p-anisaldehyde, (E,E)-α-farnesene), which explained in the mean > 92% of the total floral scents collected from plants grown at the optimum scenario (no compounds were detected from plants grown at increased temperatures; see Results), was prepared to be used for electroantennographic analyses. Given that the three bee species responded in the electrophysiological measurements to all the compounds included in this mixture (see Results), the same mixture was also used for behavioral experiments (see below). When pipetted on filter papers (diameter 3.7 cm; Whatman) used for behavioral experiments, sampled by dynamic headspace, and analyzed by GC/MS, this mixture resembled the relative and absolute composition of these compounds in the natural samples. The synthetic scent mixture was prepared with compounds available in the reference collection of the Plant Ecology lab of the Paris-Lodron University of Salzburg in the highest purity available (> 90%). The solvent to dilute the compounds and used as a negative control in behavioral experiments (see below) was acetone (Sigma-Aldrich, 99.8%).
The other compound detected in the floral scent samples were not included in the synthetic mixture as they were found only in less than half of the flower scent samples (benzyl tiglate) or were identified just after the physiological and behavioral experiments have been performed (ionones, benzyl benzoate).
2.5 Electroantennographic detection
The synthetic scent mixture was tested on the antennae of the three bee pollinators (N = 7 worker bee individuals each of A. mellifera and B. terrestris, and 5 females and 4 males of O. bicornis) by GC/EAD (gas chromatography coupled with electroantennographic detection) to evaluate the compounds eliciting antennal responses. The GC/EAD system, the same as that used by Heiduk et al. (2016), consisted of a gas chromatograph (Agilent 7890A, Santa Clara, California, USA) equipped with a flame ionization detector (FID) and an EAD setup (heated transfer line, 2-channel USB acquisition controller) provided by Syntech (Kirchzarten, Germany). A volume of 1 µl of the samples was injected (temperature of injector: 250°C) splitless at 40°C oven temperature, followed by opening the split vent after 0.5 min and heating the oven at a rate of 10°C min− 1 to 220°C. A DMT Beta SE column (30 m long, inner diameter 0.25 mm, film thickness 0.23 µm, MEGA-DEX) was used for the analyses, and the column flow (carrier gas: hydrogen) was set at 3 ml min− 1. The column was split at the end by a µFlow splitter (Gerstel, Mülheim, Germany) into two deactivated capillaries leading to the FID (2 m x 0.15 µm) and EAD (1 m x 0.2 µm) setups. Makeup gas (N2) was introduced in the splitter at 25 ml min− 1. The outlet of the EAD was placed in a cleaned and humidified airflow that was directed over the antenna of the pollinators. The antennae were cut at their base and tip, inserted between two electrodes filled with an insect ringer (8.0 g/l NaCl, 0.4 g/l KCl, 0.4 g/l CaCl2), and connected to silver wires as described previously (Dötterl et al. 2005).
A floral compound was considered EAD-active in a bee species when it elicited a depolarization response in at least four individuals.
2.6 Behavioral experiments
Behavioral assays were performed to test whether the synthetic flower scent of strawberry plants is capable of attracting strawberry-naïve bee pollinators. These assays were conducted outdoors (A. mellifera, B. terrestris) and indoors (O. bicornis) at the Paris-Lodron University of Salzburg. Tests with O. bicornis were performed indoors between May and June 2021, as weather conditions during their flight period did not allow testing outdoors.
The outdoor behavioral experiments were performed between June and August 2021 in a flight cage (wooden construction clamped with white gauze of 8 x 4 x 2.2 m) in the Botanical Garden, the same as that successfully used with bees before (Rachersberger et al. 2019). In this flight cage, we kept the bees (A. mellifera hive, obtained from a local beekeeper, with 10 combs; B. terrestris hive, obtained from Biobest®, Belgium), and there was Reseda lutea (Resedaceae) continuously flowering and used as a pollen and nectar resource. Scent samples were offered on artificial flowers in dual-choice assays, with a distance of 1.50 m between them. The artificial flowers were made of blue bond paper of 7 cm diameter and tied on wooden sticks. White filter paper of 1 cm diameter (Whatman), onto which a scent sample was applied, was placed in the middle of the artificial flower (Supplementary Information Fig. 1A and 1B).
The indoor behavioral experiments with O. bicornis (obtained from Mauerbienenzucht®, Germany) were performed in a small cage (30 x 30 x 30 cm) placed in an experimental room (temperature: 25°C) that was illuminated by five T26 EVG Grolux lamps. For each experimental run (17 runs in total), we tested 10 individuals per time (males and females separately) for one hour. Bees were fed sugar water (50%, w/w) inside the cage in a small pot. The samples were offered in dual-choice assays on filter papers (diameter 3.7 cm; Whatman) placed on aluminum foil on the bottom of the cage with a distance of 20 cm between them (Supplementary Information Fig. 1C and 1D, Video 1).
For each pollinator, we tested the synthetic scent mixture against a negative control (acetone). To test for a potential side bias, the synthetic scent mixture was also tested against itself (only for outdoor tests).
For all assays, 150µl of the synthetic mixture /acetone was used. As determined by GC/MS of headspace samples collected at ambient temperature, the absolute amount of scent offered to pollinators was equivalent to the scent of 100 flowering plant individuals, i.e., representing a small crop area (Supplementary Information Table 1). Thus, the scent offered to the bees was in a natural range, as a bee that approaches a strawberry crop field might easily be exposed to the headspace of 100 individuals. One bioassay trial lasted for 1 h, whereas the position of the artificial flowers/filter papers were changed and the scent mixture was renewed after 30 min. Bees that landed on artificial flower/filter paper were recorded and marked with a nontoxic pen (Posca® - Tokyo, Japan) to avoid counting an individual bee twice. A specific assay was replicated (one trial = one replicate) until a minimum of 15 bees responded. Overall, we performed bioassays on a total of 16 days, for in total of 38 hours (outdoor: A. mellifera − 6 days/14 hours, B. terrestris − 5 days/15 hours; indoor: O. bicornis − 5 days/9 hours).
Table 1
Total absolute amount of floral scent (mean ± standard error) and relative amount of the different floral scent compounds (%; mean ± standard error) emitted by strawberry plants (N = 10) grown under optimum temperature conditions. No scent was detected in samples collected from plants grown at increased temperatures. Compounds are listed according to chemical class. They were identified based on mass spectra and retention indices (KRI) of authentic standards, except α-ionone, which was identified based on mass spectrum and retention index of literature data.
| | Optimum |
total absolute amount of scent (ng/flower/h) | | 10.45 ± 2.29 |
Compounds | KRI | |
Aromatics | | |
benzyl alcohol | 1037 | 3.7 (± 1.1) |
methyl salicylate | 1205 | 4.3 (± 1.8) |
p-anisaldehyde | 1265 | 81.9 (± 5.8) |
benzyl tiglate | 1508 | 0.6 (± 0.3) |
benzyl benzoate | 1788 | 4.5 (± 1.8) |
Irregular terpenes | | |
α-ionone | 1436 | 2.3 (± 1.0) |
dihydro-β-ionone | 1442 | 0.6 (± 0.3) |
Sesquiterpenes | | |
(E,E)-α-farnesene | 1514 | 2.1 (± 1.4) |
2.7 Data analysis
The dual-choice behavioral experiments were analyzed by exact binomial tests of goodness-of-fit using the spreadsheet provided by http://udel.edu/~mcdonald/statexactbin.html.