Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2018, 66, 13065−13072
Honey Norisoprenoids Attract Bumble Bee, Bombus terrestris, in New Zealand Mountain Beech Forests Ashraf M. El-Sayed,*,† C. Rikard Unelius,†,‡ and David M. Suckling†,§ †
The New Zealand Institute for Plant & Food Research Limited, Gerald Street, 7608 Lincoln, New Zealand Department of Chemistry and Biomedical Sciences, Linnaeus University, SE-391 82 Kalmar, Sweden § School of Biological Sciences, University of Auckland, Tamaki Campus, Building 733, Auckland, New Zealand
J. Agric. Food Chem. 2018.66:13065-13072. Downloaded from pubs.acs.org by UNIV OF GOTHENBURG on 12/19/18. For personal use only.
‡
ABSTRACT: Three varieties of honey of different dominant floral origin were found to attract social Hymenoptera, including the large earth bumble bee, Bombus terrestris, in a New Zealand mountain beech forest. This study was undertaken to identify volatile organic compounds that induce the attraction of bumble bees to honeybee (Apis mellifera) honey. We analyzed the chemical composition of the volatile organic compounds produced in three distinct varieties of honey (i.e., manuka, honeydew, and clover honey). The composition of the chemical profile of the three honey varieties differed in the quality and in the ratio of compounds in the headspace. o-Methoxyacetophenone was the main compound in the headspace of all three honey varieties. Among the 40 compounds identified in the headspace in the three varieties, only seven shared compounds (i.e., benzaldehyde, benzyl alcohol, phenylacetaldehyde, 2-phenylethanol, isophorone, 4-oxoisophorone, and o-methoxyacetophenone) were present in the headspace of the three honey varieties. The relative attractiveness of various blends of the seven common compounds found in the three honey varieties was tested for the attraction to bumble bees in a mountain beech forest. A binary blend of isophorone and 4-oxoisophorone at a ratio of 90:10 was the most attractive blend for both bumble bee workers and queens. A small number of honey bee workers were also attracted to the former binary blend. Our study represents the first identification of a honey-derived attractant for bumble bees and honey bees. The potential application of our finding for monitoring of bumble bees or to enhance crop pollination and help to tackle the current concern of a global pollination crisis is discussed. KEYWORDS: social hymenoptera, bumble bees, honey, attractant, isophorone, 3,5,5-trimethylcyclohex-2-enone, 4-oxoisophorone, o-methoxyacetophenone
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The European Union defines honey as “natural viscous sweet substance produced by Apis mellifera (Linnaeus) bees from the nectar of plants or from secretions of living parts of plants or excretions of plant-sucking insects on the living parts of plants, which the bees collect and transform by combining it with specific substances of their own”.4 The product is then deposited in honeycombs to dehydrate, ripen, and mature. Natural honey consists of the monosaccharides fructose and glucose, the disaccharide sucrose, water, proteins, organic acids, vitamins minerals, pigments, phenolic compounds, and a large variety of volatile organic compounds.5,6 The composition, color, aroma, and flavor of honey depend mainly on the flowers, geographical regions, climate, and honey bee species involved in its production and are also affected by weather conditions, processing, and manipulation.7,8 The volatiles from honey are known to vary widely with the floral origin, and honey headspace has been investigated mainly to determine the authenticity of honey, where unifloral honey is more valuable than multifloral honey. Over 200 compounds have been identified in the headspace of honey originating from different geographical locations.9 Since honey bees make honey from nectar collected from flowers, it is expected that many honey volatiles will be of a
INTRODUCTION The mountain beech forests (Nothofagus spp.) are the largest remaining indigenous forest type in New Zealand and cover one-quarter of New Zealand (ca. 1.2 million ha). These beech forests have become a home to various invasive hymenopteran species including social wasps (two Vespula species) and bumble bees Bombus species at forest margins. These two invasive insect groups are thriving in association with the beech forest mainly because of the abundant carbohydrate-rich honeydew and the lack of natural enemies. Bumble bees were brought from England to New Zealand by settlers, mainly for red clover pollination.1 Since their introduction, bumble bees have spread all over New Zealand and to some offshore islands. Four species of bumble bees occur in New Zealand, including the two short-tongued Bombus terrestris (Linnaeus) and Bombus subterraneus (Linnaeus) and the two long-tongued Bombus ruderatus (Fabricius) and Bombus hortorum (Linnaeus). B. terrestris is the most abundant among the four bumble bee species. Bumble bees, especially B. terrestris, are important pollinators of pastoral legumes. They are efficient pollinators of flowers with a widely separated style and stamens, notably kiwifruit (Actinidia chinensis), passion fruit (Passif lora edulis), feijoa (Acca sellowiana), and cucurbits (Cucurbita spp.).2 The introduction of bumble bees for pollination in New Zealand was the first successful introduction of this genus in the world, and this population recently acted as a Noah’s Ark for the United Kingdom population after its extinction.3 © 2018 American Chemical Society
Received: Revised: Accepted: Published: 13065
August 5, 2018 October 18, 2018 November 11, 2018 November 12, 2018 DOI: 10.1021/acs.jafc.8b04175 J. Agric. Food Chem. 2018, 66, 13065−13072
Article
Journal of Agricultural and Food Chemistry floral origin, while others will be the result of biochemical reactions of the compounds present in the nectar. Unlike honey bees, bumble bees store small quantities of nectar in waxy cells which are called honeypots. Both bumble bees and honey bees utilize floral volatiles to locate flowers for nectar collection. On the other hand, interactions between honey bees and other insects are naturally occurring phenomena, for example, some insect species are pests in beehives, e.g., the small hive beetle, Aethina tumida (Murray), the greater wax moth, Galleria mellonella (Linnaeus), and the lesser wax moth, Achroia grisella (Fabricius), whose larvae feed on honey and wax combs. Other insect species, like the German wasp, Vespula germanica (Fabricius), are known to rob weak beehives.10 In addition, honey has been used traditionally as an attractant for insects, mainly hymenopteran species.11,12 It is expected that chemical signals from honey would play a key role in these interactions. In spite of the fact that honey is produced by insects and can regulate the response of other insects seeking the rich carbohydrate source, it is surprising that no previous study has been conducted to characterize particular semiochemicals that originate from honey and affect heterospecific insect behavior. In pilot experiments conducted at the edge of the mountain beech forests, several varieties of honey were tested for the attraction of the social wasps Vespula vulgaris (Linnaeus) and V. germanica. In these field experiments, many B. terrestris workers and queens were caught in traps baited with raw honey within a short period of time. The current study was undertaken to identify volatile organic compounds in honey that mediated the attraction of bumble bees to honey. We anticipated that the results of this work might provide a honeybased volatile attractant that could be used to monitor wild bumble bee populations, enable harvest of wild queens, or enhance crop pollination. Pollinators provide a key ecosystem service to wild and cultivated plant species for fruit and seed production.13,14 With the current global changes, the recent decline of insect pollinator communities is a major concern of a global pollination crisis.15 Among pollinators, bumble bees have reportedly experienced a worldwide reduction in population density.16,17 Therefore, the development of any tool, including attractants that can be used to manipulate bee’s behavior, could be a critical tool to tackle such a pollination crisis, especially in the agricultural sector.
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was conditioned according to the instructions provided by the supplier. Honey samples were maintained and magnetically stirred for 1 h at 70 °C to allow equilibration. Sampling of the volatile honey compounds was done by inserting the sheathed fiber through the septum and exposing it to the headspace for 30 min.18 The fiber was then retracted and transferred to the injector port of the gas chromatograph where the compounds were desorbed for 5 min. Control samples were collected from the surrounding lab atmosphere to distinguish between honey volatile compounds and ambient contaminants. Gas Chromatography/Mass Spectrometry Analysis (GC/ MS). Honey samples were analyzed using a Saturn 2200 GC/MS (Varian Inc., Walnut Creek, CA, USA). The GC/MS system was equipped with a 30 m × 0.25 mm i.d. × 0.25 μm VF5-MS capillary column (Varian Inc.). Helium was used as the carrier gas at a flow rate at 1 mL min−1. The spectra were recorded at an ionization voltage of 70 eV over a mass range m/z from 20 to 499. The transfer line and the trap were held at 250 and 180 °C, respectively. Compounds were identified by comparing their mass spectra with the authentic standards and the NIST MS library as well as coincidences for Kovats retention indices published in the literature.19 Compounds to be tested were purchased, and their presence in the honey was confirmed by injection in the GC/MS instrument. Chemicals. The seven compounds that were present in all three honey types were selected for behavioral experiments. Benzaldehyde (98%), benzyl alcohol (99%), phenylacetaldehyde (90%), 2-phenylethanol (≥99%), isophorone = 3,5,5-trimethylcyclohex-2-enone (97%), 4-oxoisophorone = 2,6,6-trimethylcyclohex-2-ene-1,4-dione (98%), and o-methoxyacetophenone (99%) were obtained from Sigma-Aldrich Chemical Co. (St Louis, MS). Field Experiments. All field experiments were conducted on a matagouri, Discaria toumatou (Raoul), covered riverbed adjacent to a mountain beech forest in Hawdon Valley near Arthur’s Pass in Canterbury, the South Island, New Zealand (42°59.54′S, 171°47.60′E). Experiments were conducted from the end of February to early March. During this time, bumble bee queens will start to emerge, although the workers are still the dominant casts in bumble bee colonies and the beech forest. This will impact the number of the number of queens captured. Red delta traps made of plastic corflute with an adhesive-coated base (Suckling and Shaw 1992) were used. Five replicates of each treatment were used and arranged in a randomized block design. This design was laid out using five trap lines established at approximately 20 m intervals. Each line consisted of eight (trial 1), six (trial 2), four (trial 3), or five (trial 4) trapping stations, with a full replicate of treatments on each line. Trapping stations were placed at 20 m intervals in each trap line. Traps were positioned 1.7 m above the ground in the matagouri vegetation and were spaced 20 m apart in each row. The attractant blends were formulated in a permeable polyethylene bag (100 μm wall thickness, 20 mm × 20 mm, Masterton, New Zealand) of 100 μm wall thickness (45 mm × 50 mm) with a piece of felt (15 mm × 45 mm) inserted as a carrier substrate. The polyethylene sachets were placed in the center of the sticky base. Sticky bases were removed, and insects were identified and counted after each experiment. There were very low bycatch in all of the trials, all bumble bees caught in the traps during this study were identified as B. terrestris, and all of the honey bees were identified as A. mellifera. In all trials, traps with a dispenser without chemicals were used as the negative control. Testing Individual Compounds. This trial was conducted February 25−27, 2012 to investigate the relative attractiveness of the individual honey compounds. Lures containing each of the seven compounds in Figure 1 (i.e., benzaldehyde, benzyl alcohol, phenylacetaldehyde, 2-phenyethanol, isophorone, 4-oxoisophorone, omethoxyacetophenone) were made by dispensing 100 mg of each compound on a piece of felt inside a permeable polyethylene bag that was then heat-sealed. Optimizing the Ratio of Isophorone (Ip) and 4-Oxoisophorone (OIp). The second field trial was conducted March 1−3, 2012 to investigate the relative attractiveness of binary blends containing various ratios (100:0, 10:90, 50:50, 10:90, and 0:100 mg) of
METHODS AND MATERIALS
Honey Samples. Five replicates from five different batches of three different varieties of honey were purchased from Ecrotek Beekeeping Supplies, Christchurch, New Zealand. The three honey varieties studied were manuka from the nectar of the manuka tree, Leptospermum scoparium (J. R. Forst. and G. Forst), honeydew exuded from the scale insects Ultracoelostoma assimile (Maskell) and Ultracoelostoma brittini (Morales), and white clover Trifolium repens (Linnaeus). Ecrotek Beekeeping Supplies personnel confirmed the authenticity and the origin of the honey. All honey samples originated from New Zealand. Samples were stored at 4 °C until subjected to headspace analysis. Headspace SPME Analysis. Prior to analysis, the honey sample (5 g) was placed into a 15 mL clean glass vial for solid-phase microextraction (SPME); after adding 5 mL of distilled water, the vial was sealed with a magnetic cap with a PTFE/silicon septum and vortexed until complete homogenization was achieved. The vials were cleaned by heating them at 150 °C overnight prior to the analysis. The SPME fiber 50/30 mm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) used for sampling of honey volatiles was supplied by Supelco (Bellefonte, PA, USA). Prior to use, the fiber 13066
DOI: 10.1021/acs.jafc.8b04175 J. Agric. Food Chem. 2018, 66, 13065−13072
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(23% OIp, 3% Ip) followed by honeydew honey (9% OIp, 0.9% Ip), while manuka honey had the lowest percentage (1% OIp, 0.2% Ip). Similarly, the relative ratio of benzaldehyde, benzyl alcohol, phenylacetaldehyde, and 2-phenylethanol was higher in clover honey, followed by honeydew and manuka honey. Although linalool was present in the three varieties of honey, this compound was not included in our biological test because it was previously tested (El-Sayed Unpublished data), and it never showed any behavioral response. Testing Individual Compounds. When the seven honey compounds were tested individually, significantly more bumble bee workers were attracted to isophorone, 4-oxoisophorone and o-methoxyacetophenone than to the other test compounds or the negative control, treatment, F7,32 = 12.36, P = 0.001 (Figure 2). Isophorone and 4-oxoisophorone significantly attracted more bumble bee queens and honey bee workers than other compounds tested and negative control (bumble bee queen: Treatment, F6,28 = 4.25, P < 0.02. Honey bee worker: Treatment, F6,28 = 3.6, P < 0.04). Optimizing the Ratio of Isophorone and 4-Oxoisophorone. Changing the ratio between isophorone and 4oxoisophorone significantly affected the number of bumble bee workers captured (Figure 3). Bumble bee workers: Treatment, F5,24 = 4.52, P < 0.02. Bumble bee queens: Treatment, F5,24 = 5.6, P < 0.02. The largest number of bumble bee workers was caught in traps baited with the binary blend at a ratio of 90:10 (Figure 3). The catch of bumble bee workers in traps baited with isophorone alone and binary blends of isophorone and 4oxoisophorone at ratios of 50:50 and 10:90 was not significantly different (Figure 3). The largest number of bumble bee queens was caught in traps baited with the binary blend at a ratio of 90:10 (Figure 3). There was no significant difference in honey bee workers catch between treatments (Figure 3). Investigating the Synergistic Effect of o-Methoxyacetophenone. The addition of o-methoxyacetophenone to a binary blend of isophorone and 4-oxoisophorone did not result in a significant increase in the catch of bumble bee workers, queens, or honey bee workers in traps (Figure 4). Investigating the Synergistic Effect of the Benzene Derivatives. The addition of benzaldehyde, benzyl alcohol, phenylacetaldehyde, and 2-phenylethanol to a binary blend of isophorone and 4-oxoisophorone did not result in a significant increase in the catch of bumble bee or honey bee worker (Figure 5). In contrast, the addition of these compounds to the binary blend resulted in a significance decrease in the number of bumble bee queens caught (Figure 5). The catch of bumble bee or honey bee and bumble bee queens in binary blends containing isophorone and 4-oxoisophorone was similar to the catch in traps baited with honeydew honey (Figure 5).
Figure 1. Structures of the seven test compounds and their close biochemical relationships. isophorone:4-oxoisophorone. Binary blends were made by dispensing 100 mg of each ratio on a piece of felt inside a permeable polyethylene bag that was then heat-sealed. Investigating the Synergistic Effect of o-Methoxyacetophenone (o-MAP). The third field trial was conducted March 4−6, 2012 to investigate the effect of the addition of o-methoxyacetophenone to the optimized binary blend obtained in trial two. The relative attractiveness of the binary blend containing isophorone and 4oxoisophorone, at a ratio Ip 90:OIp 10 mg, was compared to the two ternary blends containing isophorone, 4-oxoisophorone, and two ratios of o-methoxyacetophenone (Ip90:OIp10:o-MAP10 mg and Ip90:OIp10:o-MAP30 mg). Binary and ternary blends were made by dispensing 100 mg of each blend on a piece of felt inside a permeable polyethylene bag that was subsequently heat-sealed. Investigating the Synergistic Effect of Benzene-Derived Compounds. The fourth field trial was conducted March 8−10, 2012 to investigate the relative attractiveness of three blends. Blend 1 contained benzaldehyde, benzyl alcohol, phenylacetaldehyde, 2phenylethanol, isophorone, and 4-oxoisophorone at a ratio of 15:25:30:30:90:10 mg. Blend 2 contained isophorone and 4oxoisophorone at a ratio of 90:10 mg. Blend 3 contained benzaldehyde, benzyl alcohol, phenylacetaldehyde, and 2-phenylethanol at a ratio of 15:25:30:30 mg. The ratios of compounds were selected to match the average ratio of these compounds in the headspace of the three varieties of honey. Traps baited with 1 g of honeydew honey were used as positive controls. Statistical Analysis. The variance of mean captures obtained with each compound or each honey blend was stabilized using the angular transformation of counts, and the significance of treatment effects was tested using ANOVA. Significantly different treatment means were identified using Fisher’s protected least significant difference test.20
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RESULTS Chemical Analysis of Honey Volatiles. The composition of the chemical profile of the three honey varieties varied in both the number and the type of compounds present and in the ratio of compounds in the headspace. (Table 1). The objective of the chemical analysis was not to define a marker for each honey variety but rather to find common chemicals between the diverse honey varieties as candidate compounds for our biological tests. o-Methoxyacetophenone was the main compound in the headspace of all three honey varieties. The relative ratio of this compound varied between the three varieties: manuka honey had the highest ratio (83%) followed by honeydew honey (71%) with clover honey having the lowest (34%). 4-Oxoisophorone and isophorone were also present in the headspace of all three varieties, and the relative ratio of these two compounds was highest in clover honey
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DISCUSSION Chemical analysis of the honey headspace has until now been used to determine authenticity and floral origin of the honey.9 Our study is the first to investigate honey volatiles with the objective of identification of honey-derived attractants for insects. Field testing of the seven honey compounds in New Zealand beech forest resulted in the identification of the two norisoprenoids isophorone and 4-oxoisophorone at a ratio of 90:10 as the binary blend that attracts the largest number of worker and queen bumble bees and to a lesser extent honey bees. 13067
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Table 1. Average Relative Ratios (n = 5) of Volatile Organic Compounds Expressed in a Percentage of Total VOC in the Headspace of the Three Honey Varieties As Determined by Relative Peak Heights Obtained by GC/MS Elution Profiles ratio (%) compound
RIa
benzaldehydeb benzyl alcoholb phenylacetaldehydeb lilac aldehyde isomer 1-phenylethanol linalool oxide linaloolb (E)-ocimeneb 3,7-dimethyl-1,5,7-octatrien-3-ol 2-phenylethanolb isophoroneb 4-oxoisophoroneb lilac aldehyde isomer lilac aldehyde isomer 2′-hydroxyacetophenone 1-(1,4-dimethyl-3-cyclohexen-1-yl)-ethanone 2,2,6-trimethyl-1,4-cyclohexanedioneb lilac aldehyde isomer methyl salicylateb unknown α-terpineol unknown 2,6,6-trimethyl-2,4-cycloheptadien-1-one (eucarvone) unknown ethyl phenylacetate p-anisaldehydeb 1-(2-methoxyphenyl)ethanol o-methoxyacetophenone 2-methyl-6-propylphenol unknown 4-methoxyphenethyl alcohol 1-(2,6,6-trimethyl-1,3-cyclohexandien-1-yl)-2-buten-1-one (damascenone) cis-jasmone 1-(2-hydroxy-6-methoxyphenyl)ethanone 3-(3-methylphenyl)propionic acid neryl acetone β-farnesene 5,6-dihydro-6-pentyl-2H-pyran-2-one 1,3,7,7-tetramethyl-9-oxo-2-oxabicyclo[4.4.0]dec-5-ene methyl 3,5-dimethoxybenzoate
962 1034 1046 1072 1074 1089 1099 1101 1105 1112 1121 1141 1149 1152 1158 1164 1167 1175 1187 1191 1199 1216 1225 1239 1246 1254 1276 1303 1305 1318 1367 1383 1397 1401 1430 1450 1455 1478 1485 1529
manuka honey 0.14 0.34 1.16 0.35 0.07
± 0.03 ± 0.24 ± 0.20 ± 0.09 ± 0.02
2.65 ± 0.41 0.22 ± 0.07 0.68 ± 0.14 0.21 ± 0.03 1.09 ± 0.15
honeydew honey
clover honey
0.69 ± 0.28 1.23 ± 0.26 1.13 ± 0.37
1.80 ± 0.26 2.83 ± 0.33 2.62 ± 0.37
0.56 ± 0.18 5.17 ± 1.96
5.72 ± 0.86 3.09 ± 0.37
0.39 1.65 0.92 8.83 0.43 0.88
± 0.14 ± 0.27 ± 0.24 ± 2.34 ± 0.11 ± 0.16
5.87 2.39 2.68 22.64 2.65 3.82
± 2.72 ± 0.15 ± 0.35 ± 2.48 ± 0.24 ± 0.67
0.55 ± 0.10 2.65 ± 0.27 0.69 ± 0.25 1.67 ± 0.39 0.47 ± 0.25 0.38 ± 0.19 0.39 ± 0.09 1.02 ± 0.45 1.05 ± 0.27 0.86 ± 0.55
0.41 ± 0.17 0.68 ± 0.25 3.70 1.90 82.52 1.55 0.18 0.31
± 0.71 ± 0.51 ± 1.93 ± 0.33 ± 0.08 ± 0.07
1.02 ± 0.19 70.67 ± 4.31
33.83 ± 1.30
0.93 ± 0.17 0.52 ± 0.12 0.58 ± 0.42
0.51 ± 0.13
0.68 ± 0.25 0.84 ± 0.31
1.64 ± 0.38
0.42 ± 0.19
0.66 ± 0.32 0.27 ± 0.02 1.54 ± 0.33
a
Kovats index on a DB-5 column. bCompound identity was confirmed by comparison of MS and retention time of authenticated standard; the rest of the compounds were tentatively identified by comparison with published Kovatś indices and mass spectral data.
The four benzene derivatives, benzaldehyde, benzyl alcohol, phenylacetaldehyde, and 2-phenylethanol, have been found in many varieties of honey from various geographical regions.21 These four compounds showed no biological activity when they were tested individually. However, when they were combined in a quaternary blend they attracted significantly more bumble bee workers than negative controls, although significantly less than any blend containing isophorones. The four benzene derivatives are very common floral compounds19,22 and produced from other parts of plants. They are also known to be emitted by many plants when attacked by herbivores.23 The lack of attraction to individual benzene derivatives could be due to the fact that either they do not convey useful information when presented alone or they are not properly perceived by bees due to background odor
plumes as they are common compounds produced from various parts of the plant. They might convey meaningful chemical information for bumble bees only when combined. Similarly, a complete blend of floral volatiles of Canada thistles is more attractive to various flower visitors than the individual compounds.19 Benzene derivatives could result from either direct import of flower nectar volatiles by bees or the Strecker degradation during postharvest treatment of honey or from amino acids (e.g., phenylalanine) by enzyme catalysis.24,25 Among the three most attractive compounds, o-methoxyacetophenone was the least attractive compound. o-Methoxyacetophenone is a compound unique to several varieties of New Zealand and Australia honey26 and also found in Chestnut honey.27 This might indicate that some flowers in these habitats are producing this compound. It has been 13068
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Figure 2. Mean ± SE numbers of bumble bee, B. terrestris, and honey bee, A. mellifera, captured in red delta traps baited with 100 mg of each of the seven specific honey compounds at the margins of New Zealand Nothofagus beech forests. (Top) Bumble bee workers; (middle) bumble bee queens; (bottom) honey bee workers. Treatments labeled as follows: benzaldehyde (A), benzyl alcohol (B), phenylacetaldehyde (C), 2-phenylethanol (D), isophorone (E), 4-oxoisophorone (F), o-methoxyacetophenone (G), and blank. Loading of the compounds are in milligrams. Treatments labeled with the same case letters are not significantly different (n = 5, P > 0.05). Treatments that caught no bumble bees were not included in the analyses.
Figure 3. Mean ± SE numbers of bumble bee, B. terrestris, and honey bee, A. mellifera, captured in red delta traps baited with lures containing different ratios of isophorone and 4-oxoisophorone honey at the margins of New Zealand Nothofagus beech forests. (Top) Bumble bee workers; (middle) bumble bee queens; (bottom) honey bee workers. Loading of the compounds are in milligrams. Treatments labeled with the same case letters are not significantly different (n = 5, P > 0.05). Treatments that caught no bumble bees were not included in the analyses.
reported recently as a floral compound in New Zealand’s sundew Drosera auriculata (Backh. ex Planch).28 Since there are very few analyses of the headspace of New Zealand native flowers, this compound might be more abundant as a floral compound in New Zealand flora than expected, based on its occurrence in the honey.
4-Oxoisophorone was the second most abundant compound in the headspace of the three honey varieties investigated (Table 1). In our study, this compound showed moderate biological activity compared with isophorone (Figure 2). It has been reported as a floral compound but less abundant than benzene derivatives.19,22 Recently, it was identified in the inflorescence of four stone fruits crops, Prunus spp., that 13069
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Figure 4. Mean ± SE numbers of bumble bee, B. terrestris, and honey bee, A. mellifera, captured in red delta traps baited with lures containing different binary and ternary blend of isophorone, 4oxoisophorone honey, and o-methoxyacetophenone at the margins of New Zealand Nothofagus beech forests. (Top) Bumble bee workers; (middle) bumble bee queens; (bottom) honey bee workers. Loading of the compounds are in milligrams. Treatments labeled with the same case letters are not significantly different (n = 5, P > 0.05). Treatments that caught no bumble bees were not included in the analyses.
Figure 5. Mean ± SE numbers of bumble bee, B. terrestris, and honey bee, A. mellifera, captured in red delta traps baited with lures containing two blends of benzaldehyde, benzyl alcohol, phenylacetaldehyde, 2-phenylethanol, isophorone, 4-oxoisophorone, and omethoxyacetophenone at the margins of New Zealand Nothofagus beech forests. (Top) Bumble bee workers; (middle) bumble bee queens; (bottom) honey bee workers. Loading of the compounds are in milligrams. Raw honey (1 g) was used as a positive control. Treatments labeled with the same case letters are not significantly different (n = 5, P > 0.05). Treatments that caught no bumble bees were not included in the analyses.
include cherry, plum, peach, and nectarine.29 In addition, it has been found in the headspace of many honey varieties9,21,30,31 and has been reported as an attractant for a few insect species,19 including several syrphid floral visitor species.29 However, this is the first report of the attraction of bumble bees to 4-oxoisophorone. Isophorone was the most attractive compound to bumble bee workers, and the response to isophorone alone was not
different from the best binary blend (Figure 3). The relative proportions of this compound were 0.2%, 1%, and 2.4% in the headspace of manuka, honeydew, and clover honey, respectively (Table 1). Isophorone has been found in the 13070
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Journal of Agricultural and Food Chemistry
used to manage bumble bees in recreation areas where the large number of bumble bees has led to an increasing number of humans getting stung accidentally (Dr. Ken Hughey, New Zealand Department of Conservation, personal commication) or where there is a negative ecological impact due to the presence of large populations of bumble bees as the case of the invasive bumble bees in Tasmania.40 The ecological basis underlying the response of bumble bees to honey norisoprenoids is not clearly understood. There are several explanations for this phenomena. (1) Both 4oxoisophorone and isophorone are floral volatiles, and bumble bees might be responding to these compounds on this basis. (2) Honeybee hives are abundant in the mountain beech forest for the production of honeydew honey; we have observed bumble bees trying to enter these honeybee hives, suggesting that bumble bees were trying to rob honey from beehives. (3) Bumble bees produce small quantities of honey in small honeypots made of wax, and there is no study that has investigated the chemical composition and headspace analysis of the honey produced by bumble bees. However, it is likely that the two norisoprenoids reported in this study are present in the headspace of bumble bee’s honey, and if so, the bumble bees response could be in the form of opportunistic raiding of other bumble bee nests. Semiochemicals could help to lessen the impact of the recent decline in insect pollinator communities which had resulted in a global pollination crisis through the manipulation of pollinators foraging behavior. The direct application of floral volatiles cannot be used to enhance pollination because, with the lack of reward, pollinators will quickly learn to avoid such chemical signal. Honey compounds reported in this study were attractive to honey bees, A. mellifera. Since honey bees will be in a continuous exposure to these compounds, this chemical signal will be always be associated with positive rewards. Similarly, the bumble bees, B. terrestris, would be attracted to the two isophorones assumingly present in the headspace of bumble bee honey. Therefore, these honey norisoprenoids may be used to manipulate the foraging behavior of both honey bees and bumble bees to enhance crop pollination. This could be achieved by releasing the honey-based volatiles identified in this study in the field during the flowering season using slowrelease dispensers. Still, the potential application of honey norisoprenoids to enhance crops pollination remains to be investigated. Currently, we are using the honey norisoprenoids identified to harvest a large number of bumble bee queens early in the season from populations which are thriving in the beech forests of NZ. After allowing these queens to establish nests, they can be released into cultivated areas where crop pollination is needed.
headspace of many honey varieties from different geographical regions.30−32 In addition, it has been reported as a floral compound in few plant species19,22 and has been reported to have attractive sensory properties and low odor thresholds for humans.33 Isophorone has been reported as a semiochemical compound acting as a host cue in two species pairs; it is emitted by the plant Caragana korshinskii (Kom.) and is attractive to the wood-boring beetle Chlorophorus caragana (Chevrolat).34 It is also produced by Atlantic salmon, Salmo salar (Linnaeus), and was shown to significantly attract the sea lice, Lepeophtheirus salmonis (Krøyer) (Ingvarsdottir et al., 2002). Compared with the benzene derivatives, isophorone might be a more unique and more distinguishable chemical cue for bumble bees in the mountain beech habitat and a more specific cue for carbohydrate resources. A lower threshold of perception could also help in explaining the high potency of this compound. The norisoprenoids in honey could be produced through the oxidation of isoprenoid like 3,5,5trimethylcyclo-2-hexenic structure or through the degradation of abscisic acid, a known plant hormone compound.35 Similar numbers of honey bee workers were attracted to the two norisoprenoids compounds and crude honey (Figure 5). The response of honey bee workers to crude honey is probably an innate response that forms the basis of opportunistic raiding within this species, and this is the first report of the attraction of honey bee workers to honey-derived synthetic compounds. Our results suggest that these compounds are also acting as a foraging stimulus signal for bumble bee workers. Raiding of honey bee honey also occurs in Argentine ants,36 and it is possible that these characteristic compounds are volatile cues for the ants. Both norisoprenoid compounds were extremely attractive to bumble bees, and traps can be saturated in a few hours in the mountain beech forest environment in New Zealand. However, when these compounds were tested in urban areas in New Zealand, bumble bees approached the odor sources but only a small number entered the same traps (El-Sayed, unpublished data). Odor learning in social Hymenoptera has quite commonly been achieved in the laboratory, and bees can be trained to associate an odor with a food reward,37 but this has been rarely demonstrated in nature beyond the pollinator syndrome. The attraction of bumble bees to the norisoprenoid compounds could be from associative learning of food sources that contain both compounds developed during the life span of the bumble bees in the mountain beech forest similar to associative learning developed for the honeydew odor in social wasps in beech forest.38 The ecological impact of the large earth bumble bee populations adjacent to the New Zealand mountain beech forests may have been overlooked and neglected in the shadow of the vast ecological catastrophe imposed by the predatory Vespula wasps.39 In Tasmania, bumble bees are considered to be invasive species, and because of their preference for introduced plants, this might increase the spread of many exotic weed species, where seed production and gene flow in native plants may be affected by the behavior of introduced bumble bees.40 In New Zealand, potentially native insect species that specifically feed on the same food resource as the bumble bees may locally be in competition. The identification of a potent attractant for bumble bees will enable monitoring of the bumble bee population in the mountain beech forests across New Zealand using synthetic compounds as a tool in ecological studies. In addition, this attractant could also be
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Ashraf M. El-Sayed: 0000-0003-2999-9009 C. Rikard Unelius: 0000-0001-7158-6393 Funding
This work was supported by the Ministry of Business Innovation and Employment (Contract CO6X0811) and Better Border Biosecurity (www.b3nz.org). Linnaeus University is acknowledged for financial support to C.R.U. 13071
DOI: 10.1021/acs.jafc.8b04175 J. Agric. Food Chem. 2018, 66, 13065−13072
Article
Journal of Agricultural and Food Chemistry Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Andrew Gibb, John Revell and Mailee Stanbury for technical assistance, Barry Donovan for help with the identification of trapped bumble bee species.
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DOI: 10.1021/acs.jafc.8b04175 J. Agric. Food Chem. 2018, 66, 13065−13072
