Use of honey bees (Apis mellifera L.)

Research Article Received: 20 January 2012

Revised: 4 April 2012

Accepted: 21 April 2012

Published online in Wiley Online Library: 1 June 2012

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5742

Use of honey bees (Apis mellifera L.) to detect the presence of Mediterranean fruit fly (Ceratitis capitata Wiedemann) larvae in Valencia oranges Keith Chamberlain,∗ Mathilde Briens, Jennifer H Jacobs, Suzanne J Clark and John A Pickett Abstract BACKGROUND: When fruit deteriorates a characteristic profile of volatile chemicals is produced that is different from that produced by healthy fruits. The identification of such chemicals allows the possibility of monitoring the fruit for early signs of deterioration with biological sensors. The use of honey bees and other insects as biological sensors is well known. This study aimed to identify the volatiles produced by oranges infested with larvae of the Mediterranean fruit fly and to test the ability of honey bees, conditioned to this volatile chemical profile, to detect such oranges. RESULTS: Seventeen compounds that were present in higher concentrations in the volatile profiles of infested oranges than in those of insect-free fruits were mixed at the same relative concentrations as those in the collected volatiles of infested oranges. The synthetic mixture was used to train honey bees by classical Pavlovian conditioning and subsequent tests showed that they were then able to discriminate between medfly-infested and uninfested oranges. CONCLUSION: This study demonstrates an innovative way of detecting, at an early stage, the symptoms of damage to oranges by the Mediterranean fruit fly. c 2012 Society of Chemical Industry Keywords: honey bee; Mediterranean fruit fly; orange; proboscis extension reflex; volatile markers

INTRODUCTION

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Infestation of food by insects or contamination by microorganisms can be undetectable during harvest, storage and transport to the point of use, leading to considerable food wastage. At present, there is no available technology that can detect such contamination before there are visible signs. An example of such a situation lies in the import of oranges to the UK for juice extraction and sale in the high-end supermarkets as freshly squeezed orange juice. The shipping time for oranges imported from Spain, in particular, is only 4 days, which is insufficient time for visible signs of infestation by larvae of the Mediterranean fruit fly (medfly), Ceratitis capitata Wiedemann (Diptera: Tephritidae), to develop before the fruit is processed. Consequently, some of the juice may be contaminated with whole larvae and/or larval residues when it reaches the consumer. The detection of infestation at an early stage could prevent the losses along the supply chain and the distress that such instances can cause customers. When fruit deteriorates as a result of damage, insect infestation or fungal and bacterial contamination, a characteristic profile of volatile organic compounds is produced that is different from that produced by healthy fruits.1 – 3 The identification of such chemicals would allow the possibility of monitoring the fruit for early signs of deterioration, either by chemical or biological sensors.

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The use of honey bees, Apis mellifera L. (Hymenoptera: Apiidae), and other insects as biological sensors has been widely acclaimed.4,5 The honey bee proboscis extension response (PER) is well documented6,7 and in the laboratory, the PER has been coupled with gas chromatography (GC) to pick out components, to which the bee has been conditioned, from a complex mixture of volatiles from oilseed rape flowers.8 – 10 This study describes how the volatile profile of oranges infested with medfly larvae and eggs is used to train bees by classical Pavlovian conditioning, and how the bees are then used to discriminate between healthy oranges and those that are infested with medfly larvae but are showing no visible signs.

EXPERIMENTAL Infestation of oranges Medfly pupae were placed in insect rearing cages, approximately 200 per cage, with water and food (sucrose and yeast extract

∗

Correspondence to: Keith Chamberlain, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK. E-mail: [email protected] Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK

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c 2012 Society of Chemical Industry

Use of honey bees to detect fruit flies in oranges

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powder, 4 : 1 by weight) and kept at 23 ◦ C. Adult flies emerged 2 or 3 days later and, 3 days after that, two or three washed oranges [Citrus sinensis (L.) Osbeck, Rutaceae; cv Valencia] were placed in the cage and left for 4 days. At the same time, control (uninfested) oranges were pierced with a fine needle (0.3 mm, outside diameter) 30 times and placed in an empty insect rearing cage. At the end of the experiment, when volatiles had been collected (see below), the oranges were cut open and checked for the presence of larvae.

Collection of orange volatiles An orange was placed in a 1 L glass fermentation vessel fitted with an inlet and an outlet port. Through the inlet port was sealed a 4 mm (o.d.) polytetrafluorethylene (PTFE) tube, extending to the bottom of the vessel and delivering air that had been pumped through a charcoal filter. Into the outlet port was sealed a 5 mm glass tube, 8 cm long with a slightly tapered inner end. In this tube, at the end of the tube having the constriction, was 60 mg Porapak Q (60–80 mesh) sandwiched between two plugs of silanised glass wool. Air from inside the vessel was drawn through this ‘Porapak tube’ at a flow rate of 750 mL min−1 , measured by a flow meter, for a period of 24 or 48 h. Prior to experiments, all glassware and PTFE tubing was heated in an oven at 150 ◦ C for at least 2 h and Porapak tubes were conditioned before use by washing with freshly distilled diethyl ether (1 mL) and then heating at 132 ◦ C, while passing filtered nitrogen through the tube, for at least 2 h. Volatiles were eluted from the Porapak tubes with freshly distilled diethyl ether (500 µL) and these samples were stored at −15 ◦ C until required for analysis or for bee training.

Analysis of orange volatiles The mixtures of volatiles were analysed on Agilent 6890 gas chromatographs (GCs; Agilent, Wokingham, UK) fitted with cool on-column inlets and using HP-1 (50 m ×0.32 mm, O.D. ×0.52 µm, phase thickness) and DB-WAX (30 m ×0.32 mm ×0.5 µm) columns, with hydrogen as the carrier gas at a flow rate of 2 mL min−1 . After an initial oven temperature of 30 ◦ C, the temperature was ramped up at 5 ◦ C min−1 to 150 ◦ C and then at 10 ◦ C min−1 to 250 ◦ C for the HP-1 column or 230 ◦ C for the WAX column. Detection was by flame ionisation at 250 ◦ C or mass spectrometry. For quantitative analysis, a solution of tridecane in redistilled hexane (1 µL, 10 mg mL−1 ) was added to the solution of eluted volatiles (100 µL) to act as an internal standard, and 1 µL of this mixture was injected into the GC. Relative amounts were then calculated by giving the area under a peak as a proportion of the area under the peak of tridecane (100 ng), i.e. as nanograms tridecane equivalents. Compounds tentatively identified by gas chromatography–mass spectrometry were confirmed by co-chromatography with authentic samples on both GC columns.

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Components of the mixture of 17 synthetic compounds

Compound (R/S)-α-Pinene Butyl butanoate Ethyl hexanoate Myrcene Octanal (E)-Ocimene Octan-1-ol Nonanal (R/S)-linalool (E)-4,8-Dimethylnona-1,3,7-triene Hexyl butanoate Ethyl octanoate 3-Methylbutyl hexanoate Eugenol Hexyl hexanoate (S)-(E)-Nerolidol (E, E)-Farnesol

Concentration (µg mL−1 ) 3 2 2 16 4 140 22 2 16 150 12 34 30 8 18 14 26

Honey bee conditioning and discrimination tests Honey bee collection and restraint Honey bee (Apis mellifera L., Apiidae) foragers were collected from the exit/entrance of one of the colonies maintained by Inscentinel Ltd, Harpenden, UK, and put into cages,11 30 per cage. They were given access to a paste of icing sugar and water on which to feed. After 2 h, the food was removed and the bees were starved for 16 h. The cages were then placed in a refrigerator at 4 ◦ C for about 20 min to slow the bees’ movements temporarily, for ease of handling. For training, the bees were loaded carefully into numbered bee holders (Inscentinel Ltd) that were designed to restrain them gently, whilst allowing free movement of their antennae and proboscises. Honey bee conditioning Bees were trained using the honey bee conditioning procedure first described by Bitterman et al.6 Restrained bees were held in front of a fan before and between conditioning rounds and, as needed, placed individually 0.5 cm in front of a glass tube (1 cm o.d.) from which filtered air at rate of 2 L min−1 passed across their heads and antennae. The conditioning stimulus (in this case the synthetic mixture) was introduced to the bee by diverting 10% of the air flow through a vial containing a sample of the synthetic mixture on filter paper. Training comprised five conditioning rounds during each of which a bee was placed in the clean airflow for 15 s (Fig. 1a) before presenting air containing the volatile synthetic mixture to the bee for 3 s. At the same time, the bee’s antenna was touched with a cotton bud dipped into 40% sucrose solution (Fig. 1b) to elicit a PER in which the bee extends its proboscis in preparation for feeding (Fig. 1c). As soon as this was observed, the bee was fed using the cotton bud for 3 s (Fig. 1d). In the later rounds, as bees learned to associate the synthetic mixture with a sugar reward, they would exhibit a PER spontaneously upon presentation of the conditioning stimulus. When this occurred, the bee was immediately fed for 3 s without the need for antennal stimulation. To create the stimulus sample for each conditioning round, a small amount of the synthetic mixture was placed onto a strip


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Synthetic mixture of volatiles of infested oranges A solution, in redistilled hexane, of the 17 synthetic compounds listed in Table 1 was prepared so that they were present at the stated concentrations which corresponded to their natural relative concentrations in the headspace of infested oranges. These compounds were present in larger amounts in the volatiles from infested oranges than in those from uninfested fruits.

Table 1.

www.soci.org (a)

(b)

(c)

(d)

The ability of bees to detect medfly infestation was tested by dividing each group of 18 bees (i.e. the 15 that had gone through the conditioning process and the three that were unconditioned) into two sets and exposing each set to infested and uninfested entrainment solutions in turn. The choice of solution for the initial exposure for each set was made randomly within each experimental group, e.g. in any group, one half of the bees were exposed initially (Test 1) to the infested entrainment followed by (Test 2) the uninfested entrainment, and the other half were exposed initially to the uninfested entrainment followed by the infested entrainment. After presentation of the entrained volatiles, the response of all bees to the conditioning stimulus (the synthetic mixture) was retested. Any bees that did not respond were considered to be ‘de-conditioned’ and were excluded from the data analysis.

Figure 1. Honey bee conditioning showing a bee’s head from above (a) at rest in a clean airflow; (b) an antenna is touched with sucrose solution as the conditioning stimulus is introduced into the airflow for 3 s; (c) the proboscis is extended; and (d) the bee is rewarded with sucrose solution.

of filter paper approximately 5 mm ×25 mm. The filter paper was left for 30 s for the hexane to evaporate, before being placed into the vial through which 10% of the main airflow was diverted for delivery to each bee. For the first three conditioning rounds 2 µL of the synthetic mixture was used and for the last two rounds only 1 µL was used. It should be noted that the evaporation rates from the filter paper for the 17 compounds are not all the same and, as a consequence, the relative concentrations in the air reaching the bees are not exactly the same as in the original solution. During training, bees received one blank presentation of 2 µL of hexane on a piece of filter paper in a vial. Any bees that responded were considered to have ‘learned’ the solvent or the mechanical stimulation of the switching of airflows and were excluded from the data analysis.

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Testing discrimination between infested and uninfested oranges by honey bees On three separate days, two groups of bees were conditioned to the synthetic mixture and their ability to discriminate between the volatiles collected from oranges infested with medfly eggs and larvae, and those from uninfested oranges, was tested. Each group of honey bees represented one experiment. In each group, 15 bees were conditioned to the synthetic mixture and three bees were fed in front of the fan only, with the same timing as conditioned bees, remaining untrained to serve as controls. Samples of entrainments of volatiles from oranges infested with medfly, and uninfested oranges, were prepared for testing in a similar way to the conditioning stimulus using 1 µL of the solution.


K Chamberlain et al.

Data analysis The level of acquisition of the synthetic mixture (proportion of honey bees out of the total tested that were successfully conditioned by the end of the fifth conditioning round) was compared amongst the six groups using a generalised linear model with Poisson error and log link (maximum likelihood chisquared test). The results of the discrimination tests for the six groups were combined into a single contingency table classified by the sequence in which the two treatments were applied and outcome; i.e. the bee responded in neither test or both tests (no discrimination) or in the first or second test only (discrimination). The combined data were analysed using methods described previously;12 in particular, the presence of a direct treatment effect was tested using the Mainland–Gart test (incorporating a standard Pearson’s chi-squared test) based on the subset of bees that exhibited discrimination.

RESULTS Identification of orange volatiles Typical gas chromatograms of the volatiles collected from medflyinfested and uninfested Valencia oranges, together with an internal standard (tridecane, 100 ng), are shown in Fig. 2. The numbered peaks are those that have been identified in the majority of chromatograms. Their identities are shown in Table 2, together with relative concentrations in the entrainment solution of Fig. 2. The following compounds were also identified in a large number of samples: α-pinene, myrcene, octanal, nonanal and eugenol. The two major peaks in the gas chromatograms of the headspace from both infested and uninfested oranges are the monoterpene, limonene (Fig. 2, peak 4), and the sesquiterpene, valencene (peak 17). Ethyl octanoate (13) is also present in relatively large amounts in both although the other esters, 3-methylbutyl hexanoate (14), hexyl hexanoate (15) and hexyl octanoate (20) are at higher relative levels in the infested than the uninfested samples. Compounds showing the biggest differences are (E)-ocimene (6) and (E)4,8-dimethyl-1,3,7-nonatriene (10). Almost all of the compounds identified in the headspace of oranges, infested and uninfested, are either esters of butanoic, hexanoic and octanoic acids (2, 3, 9, 11, 12, 13, 14, 15, 20) or terpenes and terpenoids (4, 5, 6, 8, 10, 16, 17, 18, 19, 21, 22, 23). Discrimination between infested and uninfested oranges by honey bees Acquisition of the synthetic mixture was similar across groups of bees (range 86–100%; average 92%, N = 88; χ52 = 6.62, P = 0.251;


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Use of honey bees to detect fruit flies in oranges

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counts

17

4 13

6

200000

150000 16 3 10

100000

15

I.S.

18 19

50000

7

8 9

12 11

1 2

20 21

14

22

5

0

-50000

-100000

-150000

-200000

10

15

20

25

30

35 min

Figure 2. Gas chromatograms of Valencia orange volatiles (A: infested, B: uninfested) on a HP-1 column with tridecane as internal standard (I.S).

Fig. 3). Eighty-eight bees underwent testing (two out of 90 bees died during training). Data from 73 out of 88 bees were analysed, with data from 15 bees excluded for the following reasons: four responded to the blank test, four had not learned and seven did not respond to the original conditioning stimulus after testing with the entrainments and were considered to be ‘deconditioned’. Of the 73 bees included in the analysis, 78% exhibited perfect discrimination (responded in only one of their two tests), and in all of these 57 cases the single PER was in response to oranges infested with medfly (χ12 = 57.0, P < 0.001; Table 3). No unconditioned bees responded to either entrainment or the synthetic mixture.

DISCUSSION

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Figure 3. Learning curves of six groups of bees trained to a synthetic 17-compound mixture for five rounds.

differences were observed even between oranges infested with P. digitatum and those infested with P. italicum. In these studies, all using Navel oranges, there were changes in the relative amounts of various terpenes, including α-pinene, myrcene and limonene, and esters such as ethyl butanoate, methyl hexanoate and ethyl octanoate, some of which were found in the present study. One of the major compounds identified here in medfly-infested and in uninfested Valencia oranges, valencene, was not found in either of those earlier investigations though it was shown to occur in the headspace of juice obtained from Navel oranges.13



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Honey bees learned rapidly to respond to a synthetic 17compound mixture, the composition of which was based on the profile of volatiles released by Valencia oranges infested with medfly larvae. The compounds in the mixture were those that were present in larger relative amounts in the volatiles of infested oranges than in the profile from uninfested oranges. After only three rounds of training, the average number of honey bees responding across the six groups was 82% and, after five rounds, this figure increased to 92%. At that time, the honey bees were able to discriminate between oranges infested with medfly larvae, but showing no visible signs of infestation, and uninfested oranges. This approach may prove useful for the detection of food spoilage caused by insects, fungi or bacteria and from non-biotic origins such as freezing. Differences in volatile emissions of healthy oranges and those damaged by common post-harvest Penicillium infections1 or by freezing3 have been investigated previously. In the former case,

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Table 2. Compounds identified in the volatiles from control and insect-contaminated oranges, with relative rates of emission∗ (i.e. ng tridecane equivalents orange−1 h−1 ) Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Compound

Control oranges

Contaminated oranges

6-Methyl-5-hepten-2-one Butyl butanoate Ethyl hexanoate (R/S)-Limonene (Z)-Ocimene (E)-Ocimene Octan-1-ol (R/S)-Linalool Methyl octanoate (E)-4,8-Dimethyl-1,3,7-nonatriene Butyl hexanoate Hexyl butanoate Ethyl octanoate 3-Methylbutyl hexanoate Hexyl hexanoate (1R, 9S)-(E)-Caryophyllene Valencene α-Panasinsen (3S)-(E)-Nerolidol Hexyl octanoate (−)-Caryophyllene oxide (E, E)-Farnesol (R/S)-α-Pinene Nonanal

0.41 1.25 17.5 36.7 0.62 5.42 1.25 2.29 1.04 5.62 5.62 3.12 55.6 1.04 11.04 11.9 108.7 6.87 1.46 2.29 2.92 1.04 – –

2.08 1.25 29.4 181.4 3.34 62.9 13.7 14.8 2.08 41.8 12.1 11.9 86.9 8.96 33.3 33.7 288.3 21.2 14.6 6.67 8.33 3.12 – –

This work formed part of a project funded by the Biotechnology and Biological Sciences Research Council (BBSRC) under the Department of Environment, Food and Rural Affairs (Defra) Food LINK programme. The industrial partners, all from UK, were Orchard House Foods Ltd, Marks and Spencer Plc, Greencell Ltd, A1 Fruit Ltd, and KG Growers Ltd. Medfly pupae were kindly supplied by Oxitec Ltd, Oxford, UK.

REFERENCES

GC peak areas from the analysis of volatiles exemplified in Fig. 2 are compared with that of the internal standard, tridecane (100 ng), to give a notional amount collected by entrainment of an orange for 24 h.

Table 3. Proboscis extension responses (PERs) of 73 bees (combined over six groups) classified by the sequence in which the two treatments∗ were applied and by the outcome in each of their two successive tests† Outcome in two successive tests

Sequence 1 (N, I) Sequence 2 (I, N) Total

Discriminating bees

Non-discriminating bees

(0,1)

(1,0)

(0,0)

(1,1)

Total

27 0 27

0 30 30

3 1 4

8 4 12

38 35 73

∗ N = uninfested (control) oranges; I = oranges infested with Mediterranean fruit fly. † 0 = no PER observed; 1 = PER observed; so that, for example, (0,1) = no PER in Test 1, PER in Test 2.

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The presence of (E)-ocimene and (E)-4,8-dimethyl-1,3,7nonatriene in greater relative amounts in the volatiles of infested than in uninfested oranges is interesting as both compounds are associated with insect damage to the vegetative parts of a number of plants.14,15 This seems to be the first observation of these particular compounds in the headspace of fruits. The training of bees to detect infestation is a relatively simple process once the relevant marker compounds have been


identified. These results could open new routes for innovative ways to detect key components of the volatile mixture of chemicals that is specific to a particular contamination or spoilage in the food industry.

ACKNOWLEDGEMENTS

∗

Sequence and treatment

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J Sci Food Agric 2012; 92: 2050–2054