Kiwifruit Flower Odor Perception and Recognition by Honey Bees

May 30, 2015 - Honey bee (Apis mellifera) perception of the flower VOCs was tested using gas chromatography coupled to electroantennogram detection...
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Kiwifruit Flower Odor Perception and Recognition by Honey Bees, Apis mellifera Andrew M. Twidle,* Flore Mas, Aimee R. Harper, Rachael M. Horner, Taylor J. Welsh, and David M. Suckling The New Zealand Institute for Plant & Food Research Ltd., Private Bag 4704, Christchurch Mail Centre, Christchurch 8140, New Zealand ABSTRACT: Volatile organic compounds (VOCs) from male and female kiwifruit (Actinidia deliciosa ‘Hayward’) flowers were collected by dynamic headspace sampling. Honey bee (Apis mellifera) perception of the flower VOCs was tested using gas chromatography coupled to electroantennogram detection. Honey bees consistently responded to six compounds present in the headspace of female kiwifruit flowers and five compounds in the headspace of male flowers. Analysis of the floral volatiles by gas chromatography−mass spectrometry and microscale chemical derivatization showed the compounds to be nonanal, 2phenylethanol, 4-oxoisophorone, (3E,6E)-α-farnesene, (6Z,9Z)-heptadecadiene, and (8Z)-heptadecene. Bees were then trained via olfactory conditioning of the proboscis extension response (PER) to synthetic mixtures of these compounds using the ratios present in each flower type. Honey bees trained to the synthetic mixtures showed a high response to the natural floral extracts, indicating that these may be the key compounds for honey bee perception of kiwifruit flower odor. KEYWORDS: Actinidia, (6Z,9Z)-heptadecadiene, (8Z)-heptadecene, (3E,6E)-α-farnesene, proboscis extension response, learning, pollination

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food sources.20,21 The exchange of nectar by trophallaxis between workers during the waggle dance is thought to be a method of floral odor communication by returning nectar foragers in the recruitment process.22 Pollen forager recruitment has received less attention in the literature, but it is likely that floral odor cues adhering to the body and pollen loads of returning foragers are used.23 In the laboratory, odor stimulation with a sugar reward is the basis for proboscis extension response (PER) training.24 Here, honey bees are trained by Pavlovian type conditioning to extend their proboscis when a sugar reward is associated with an odor cue,25 eventually giving the PER in the absence of the sugar reward. The aim of this work was to identify the floral volatiles used by honey bees to recognize kiwifruit flowers. Here we report the collection, electrophysiological testing, identification, synthesis, and behavioral testing of these odors with the goal that a better understanding of the cues used by honey bees to detect kiwifruit flowers can be used to improve pollination of this crop.

INTRODUCTION The kiwifruit (Actinidia spp.) vine, originally from China, is now an important commercial crop in several countries, including Italy, New Zealand (NZ), and Chile.1 Kiwifruit is a dioecious plant that relies on insect and wind pollination over a 2−6 week period each season.2,3 Production of marketable kiwifruit crops in NZ relies on commercial beekeepers supplying honey bee (Apis mellifera) colonies for pollination, with some growers also using artificial pollination. Full pollination and optimum fruit size typically require >1300 seeds per fruit, which equates to about 40 bee visits per flower.4 Therefore, to achieve sufficient pollination, kiwifruit orchards require a high density of beehives per hectare (8/ha recommended in NZ) compared with other commercial crops (typically 2−8 hives/ha).5,6 Kiwifruit flowers do not produce nectar, although both male and female flowers produce pollen that is attractive to honey bees.7 The lack of nectar reward and the high fruit and seed set requirements contribute to the challenge of kiwifruit pollination. The attraction of honey bees to kiwifruit flowers7,8 and floral volatiles produced by kiwifruit flowers has been previously reported.9−12 However, to our knowledge no one has identified the chemical odor cues in the floral mixture that honey bees use to perceive kiwifruit flowers. Honey bee odor perception has been studied for other crops including sunflower,13 alfalfa,14 and seven Brassicaceae15,16 crops. Where it has been studied, flowers produce a range of volatile organic compounds (VOCs), and honey bees detect only some of these compounds.13−16 Behavioral testing of honey bee responses to complex volatile mixtures suggests honey bees use only a few compounds to recognize the full floral mixture.17 Honey bees have been shown to use olfactory cues to identify food sources18,19 and when recruiting more foragers to © XXXX American Chemical Society

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MATERIALS AND METHODS

Chemicals. Solvents, reagents, and synthetic standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. The standards used for identification of kiwifruit floral volatiles had chemical purities as follows: nonanal (95%), 2-phenylethanol (99%), 4-oxoisophorone (98%), and 8-heptadecene (96%, 85% Zisomer). (3E,6E)-α-Farnesene was not available in high purity from commercial sources, so we used the method of Murray26 to extract it Received: March 5, 2015 Revised: May 20, 2015 Accepted: May 30, 2015

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DOI: 10.1021/acs.jafc.5b01165 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. GC-MS chromatograms of kiwifruit floral volatiles from pooled male flower and pooled female flower headspace. The numbered peaks correspond to the antennally active compounds. floral volatiles were recorded using GC-EAD. Honey bees were collected as they left the hives at Plant & Food Research’s Lincoln (NZ) apiary (43°38′20.99″ S, 172°28′19.53″ E) with whole head mounts (or alternatively excised antenna) used for recordings. The freshly excised head (or antenna) of an anesthetized honey bee (n = 10 for female flower, n = 8 for male flower) was mounted between silver electrodes containing either salt solution or Spectra 360 electrode gel (Parker Laboratories Inc., Fairfield, NJ, USA). One hundred microliters of each female flower extract was taken, pooled, and then concentrated 10 times under a stream of argon; this was then repeated for the male flowers. The concentrated, pooled sample was then injected into a Varian 3800 GC (Varian, Walnut Creek, CA, USA) coupled to a Syntech EAD recording unit (Syntech Research and Equipment, Hilversum, The Netherlands). Injections were splitless for 0.6 min, and helium was used as a carrier gas with a constant flow of 1 mL/min. A 1:1 split of the column effluent to the antenna and flame ionization detector (FID) was maintained for the duration of the experiment. The GC was equipped with a DB-5 ms column (J&W Scientific, Folsom, CA, USA), 30 m × 0.25 mm i.d. × 0.25 μm film thickness. The injector temperature was 250 °C, and the column oven was programmed from 40 °C (held for 2 min) to 280 °C at 10 °C/ min. The transfer line to the antennal preparation was maintained at 250 °C. Gas Chromatography−Mass Spectrometry (GC-MS). Volatile collections, reaction products, and synthetic compounds were analyzed using a Varian 3800 GC coupled to a Saturn 2200 MS (Varian). Injections were splitless for 0.6 min, and a constant flow of 1 mL/min

from apple skins, obtaining 98% chemical purity. (6Z,9Z)Heptadecadiene was also not commercially available, so we synthesized it from linoleic acid (99%) using reductive radical decarboxylation.27 (8Z)-Heptadecene was also synthesized from oleic acid (99%) according to this method to give 94% isomeric purity by GC. Headspace Sampling of Kiwifruit Flowers. Actinidia deliciosa flowers from male (n = 6) and female (n = 10) ‘Hayward’ vines were individually encased in situ on the vine using custom-made two-piece cylindrical glass chambers (radius = 20 mm, length = 60 mm). The glassware was conditioned in a hot air oven at 150 °C overnight before use. The chamber was joined around the flower, with the stem passing through a small groove in the glass that was then gently sealed with polytetrafluoroethylene tape to avoid any damage to plant tissue. A constant flow rate of 500 mL/min was used, with air entering the chamber through a charcoal filter and leaving the chamber by passing through 60 mg of Tenax#-GR 35/60 (Grace Davison Discovery Sciences, VIC, Australia). The Tenax was conditioned at 250 °C for 3 h under nitrogen before use. Controls were an empty chamber (n = 2) and a chamber containing a leaf (n = 2) set up as outlined above for male and female flowers. Two-hour volatile collections were made between 11:00 a.m. and 1:00 p.m. in a Waikato NZ kiwifruit orchard (37°44′38.12″ S, 175°38′45.49″ E) when large numbers of bee foragers were flying. The Tenax was then eluted with 1 mL of hexane and stored at −80 °C until analysis. Gas Chromatography−Electroantennogram Detection (GCEAD). Honey bee antennal depolarizations in response to kiwifruit B

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Table 1. Summary of Honey Bee Electroantennogram Responses to Volatiles Released from Male and Female Kiwifruit Flowers compound showing antennal activity nonanal 2-phenylethanol 4-oxoisophorone (3E,6E)-α-farnesene (6Z,9Z)-heptadecadiene (8Z)-heptadecene a

Kovats index DB-5 ms (DB-wax) 1105 1111 1144 1503 1669 1678

(1404) (1927) (1708) (1758) (1765) (1722)

response to male flowers (mV) −1.8 ± 1.0 −2.7 ± 0.6

response to female flowers (mV) −1.7 −3.0 −1.5 −1.5 −3.4 −3.1

−1.2 ± 0.5 −1.4 ± 0.3 −1.6 ± 0.2

± ± ± ± ± ±

0.2 0.4 0.3 0.4 0.9 0.7

male flower extract (ng)a

female flower extract (ng)a

4 62 trb 79 104 155

4 25 3 111 142 169

Estimation of quantity of volatile perceived at the antenna based on comparison with external standards. btr, trace. −CH3), 0.88 (t, 3H, J = 7.13 Hz, −CH3); 13C NMR (125.8 MHz, CDCl3) δ 130.20 (C6 and C10), 127.96 (C7 and C9), 31.88 (C3), 31.54 (C15), 29.70 (C12), 29.36 (C4), 29.30 (C13), 29.23 (C14), 27.25 (C5 or C11), 27.21 (C11 or C5), 25.64 (C8), 22.68 (C2), 22.58 (C16), 14.10 (C1 or C17), and 14.06 (C17 or C1); MS data 70 eV, m/z (relative intensity) 236 (9, M+), 152 (3), 138 (7), 124 (11), 109 (25), 95 (55), 81 (78), 67 (100), 55 (26), and 41 (37). Proboscis Extension Response (PER). The general methodology based on Pavlovian conditioning and reviewed by Matsumoto et al.25 was followed. The synthetic flower mixtures used for PER were made up of the six antennally active compounds at the ratios present in the natural flower extracts (Figure 1). 1-Nonanol was chosen as the untrained control compound as it is readily detected by honey bees over a range of concentrations31 (from ng to mg) and is a standard control compound for PER experiments.25 Honey bees were collected in the morning and harnessed in small cylindrical tubes of 8 mm i.d. Bees were then fed 4 μL of 50% sucrose solution and left for 3 h before training. Bees were set 5 cm in front of a syringe containing 1 μg of a synthetic mixture from either male or female kiwifruit flowers deposited on filter paper. Bees had a 15 s settling time before being exposed to 30 mL of air containing the odor over 4 s. This corresponds to the conditioned stimulus (CS+). After 3 s of odor exposure, bees’ antennae were gently touched with a toothpick soaked in 50% sucrose solution and once the proboscis was extended in response, a sugar reward was given corresponding to the unconditioned stimulus (US). The association in time of the CS+ with the US is expected to trigger the formation of a memory of the odor cues with the presence of the reward. Each bee was trained four times, with 10 min intervals between training. Bees were rested for 1 h before testing in a random order to three odor blends: 1 μg of the synthetic flower mix on which they were trained, 1 μg of the correspondingly sexed flower extract, and an untrained control compound, 100 μg of 1-nonanol. In the absence of sugar reward, bees’ responses to the presentation of each odor were recorded as 1 if they showed a PER or as 0 if they did not show a PER. If bees responded positively to the synthetic flower mix as well as the natural extract, we considered that this was a demonstration that the synthetic flower mix was perceived as similar to the natural flower extract. The ability of bees to discriminate between the male or female flower extract when trained on the synthetic mix of either sex was also tested. Statistical Analysis. In total, 75 bees were trained with the PER. Thirty-seven bees were trained and tested with the kiwifruit female flower synthetic mix, and 38 bees were trained and tested with the kiwifruit male flower synthetic mix. Among those, seven bees were excluded from the final analysis because of death or lack of hunger. A chi-square test was used to compare the observed proportions of bees responding or not to the odors for each test to the calculated expected values if the null hypothesis of independency was assumed. p values of 100 volatile components, with up to 24 compounds giving EAD response but only 4 compounds were detected by 100% of the bees.13 The seven Brassicaceae crops tested by Kobayashi et al.15 contained 52 compounds across the species, but only 14 compounds were physiologically active. Blight et al.16 found 16 behaviorally active compounds in Brassica napus. Of these active compounds, kiwifruit shared 2-phenylethanol and D

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(3E,6E)-α-farnesene with some of the Brassicaceae species and none with the sunflower, whereas sunflower and Brassicaceae shared 1,8-cineole and β-elemene as bee-active compounds. Honey bees have evolved over millions of years alongside angiosperms, acting as generalist pollinators for a multitude of flowers in many different environments. It does not come as a surprise to find that they are cueing in on only some of the floral compounds in a particular flower species and that there are common cues between plant species.35 Nonanal, 2phenylethanol, 4-oxoisophorone, and (3E,6E)-α-farnesene are all well-reported plant volatiles and insect attractants.35 (8Z)Heptadecene is reported as a plant volatile for boronias36 and blue roses37 as well as an attractant across many insect orders.35 To our knowledge this is the first report of (6Z,9Z)heptadecadiene as a floral compound, but it is previously reported as a VOC from astigmatid mites, oribatid mites, and geometrid moths.35 The large EAD response by honey bees to (8Z)-heptadecene and (6Z,9Z)-heptadecadiene may not be adapted from a floral origin. (8Z)-Heptadecene has been reported as a compound associated with bee larvae,38 nurse bees,39 and pollen foragers,40 but its behavioral function is not yet known. (6Z,9Z)Heptadecadiene may also be a honey bee-produced compound, but remains to be identified as such for Apis mellifera. Nonetheless, (6Z,9Z)-heptadecadiene provides an excellent species-specific floral odor for training bees to kiwifruit flowers. Honey bees trained to the synthetic floral mix responded similarly to the natural extract, indicating that recognition of kiwifruit flower odor may be reliant on only a few key odor compounds of the floral headspace. This is in line with the work of Reinhard et al.,17 who showed that honey bees learn a few key compounds in recognition of complex volatile mixtures. In addition, honey bees did not seem to discriminate between male and female flower extracts when trained to either floral sex synthetic mix. Differences in visitation rate between male and female flowers reported by Goodwin and Steven7 cannot be explained by odor cues, but instead may be based on visual cues potentially including the abundance of pollen in the male flower. Several field trials have been carried out to encourage honey bees to visit more kiwifruit flowers, but with little success.41,42 In NZ kiwifruit orchards, Goodwin et al.43 have shown that providing in-hive feeders of sugar solution during the flowering period increases the amount of kiwifruit pollen collected by those hives. However, kiwifruit still need high hive densities to achieve sufficient pollination rates, and further behavioral manipulations at the colony level will be required for more efficient pollination. This study has highlighted that only a few odors from kiwifruit flowers are likely to be implicated in bee foraging behavior. A better understanding of how honey bees perceive and respond to floral odors at the hive level will be needed before any behavioral manipulation to improve pollination can be undertaken, but this study has enabled the next step in this process.

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This work was funded by the New Zealand Ministry of Business Innovation and Employment (CONT-33481-BTRPFR). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Barry Bunn for running NMR on the diene. We also thank Mark Goodwin and Rikard Unelius for their valuable comments on the manuscript.

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REFERENCES

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*(A.M.T.) E-mail: [email protected]. Phone: +64-3-9777347. Fax: +64-3-3252074. E

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DOI: 10.1021/acs.jafc.5b01165 J. Agric. Food Chem. XXXX, XXX, XXX−XXX