Margaret M. Blight

Identification of Floral Volatiles Involved in Recognition of Oilseed Rape Flowers, Brassica napus by Honeybees, Apis mellifera

Volatiles from oilseed rape, Brassica napus, flowers were sampled by air entrainment and their relevance to the natural odor profile of the flowers was confirmed by conditioned proboscis extension (CPE) assays with honeybee, Apis mellifera L., foragers. Coupled gas chromatography (GC)-CPE analysis of the air entrainment samples was used to locate key compounds involved in the recognition of B. napus flowers, and the compounds were then identified using coupled gas chromatography-mass spectrometry and comparison with authentic samples. Six regions of the gas chromatograms elicited CPE responses from bees previously conditioned to the total extract, and from these areas 16 compounds were identified that elicited CPE activity from conditioned bees when tested with synthetic samples. Eight of the 16, α-pinene, phenylacetaldehyde, p-cymene, α-terpinene, linalool, 2-phenyl-ethanol, (E,E)-α-farnesene, and 3-carene, gave the highest responses. When the bees were conditioned to the total extract of flower volatiles, a mixture of the eight components elicited responses from 83% of the individuals, suggesting that the eight-component mixture accounted for a major part of the CPE activity of the total extract. In addition, a mixture of the three most active compounds, phenylacetaldehyde, linalool, and (E,E,)-α-farnesene, evoked responses from 85% of the bees after the latter had been conditioned to the eight-component mixture. Thus, these three compounds appear to play a key role in the recognition of the eight component mixture and, by inference, of oilseed rape flowers.

INFLUENCE OF VISUAL CUES AND ISOTHIOCYANATE LURES ON CAPTURE OF THE POLLEN BEETLE, Meligethes aeneus IN FIELD TRAPS

The effect of trap design, trap color, and isothiocyanate (NCS) lures on the capture of the pollen beetle, Meligethes aeneus, was studied in field experiments. Unbaited yellow water and adhesive traps were equally attractive. A small sticky card trap with a yellow upper face, mounted at 45° to the vertical, was attractive throughout the year, but horizontal yellow cards were attractive only in spring, and vertical cards only in autumn. Yellow–green and white water traps were less attractive than yellow, while grass green, cream, and black were unattractive. M. aeneus was attracted equally to four alkanyl, three alkenyl, and to 2-phenylethyl isothiocyanate lures, when the release rates were in the range of 5–30 mg/day. Attraction to the most effective unbaited yellow traps was enhanced 1.7–3.3 times with the addition of a lure comprising a mixture of allyl, 3-butenyl, 4-pentenyl, and 2-phenylethyl NCS. Interactions were observed among the visual and odor cues. A yellow sticky card trap, baited with 2-phenylethyl NCS and mounted at 45° to the vertical, may be used throughout the year to monitor M. aeneus.

RECOGNITION OF COMPLEX ODORS BY RESTRAINED AND FREE-FLYING HONEYBEES, Apis mellifera

Complex odor recognition in the honeybee was investigated using two behavioral assays: (1) the conditioning of the proboscis extension (CPE) with restrained individuals, and (2) the observation of foragers visiting an artificial feeder in a flight room. Nine compounds, previously identified as oilseed rape flower volatiles, were tested either individually or in mixtures. Different sets of experiments were done to determine: (1) the acquisition rate of the nine compounds in the CPE assay, and (2) the discrimination of the individual compounds after conditioning to a mixture, using the CPE assay and free-flying foragers. After conditioning to a complex mixture, honeybees established a hierarchy among the components, with some of them accounting for a major part of the behavioral activity of the mixture. Both behavioral assays led to the same classification of compounds, indicating good agreement between discriminating abilities of restrained individuals and of a population of foragers. The key compounds for recognition of these mixtures were those that were well learned when presented individually. However, the recognition of some compounds was affected by the other components of the mixture, with the activity of some compounds being either enhanced or reduced.

RESPONSE OF THE POLLEN BEETLE, Meligethes aeneus, TO TRAPS BAITED WITH VOLATILES FROM OILSEED RAPE, Brassica napus

The response of the pollen beetle, Meligethes aeneus, to yellow water traps baited with individual lures of 25 floral volatile compounds was studied in 17 field experiments. The compounds comprised seven nitrogenous amino acid derivatives, five nonnitrogenous amino acid derivatives, nine fatty acid derivatives, and four isoprenoids. Twenty compounds affected the trap catch of M. aeneus, but response was often dependent on release rate. Most compounds were attractive, but four fatty acid derivatives were repellent. 1-Hexanol was either attractive or repellent, depending on the release rate. It is suggested that M. aeneus responds to this large number of chemically diverse compounds because it is polyphagous.

Effect of visual cues and a mixture of isothiocyanates on trap capture of cabbage seed weevil, Ceutorhynchus assimilis.

The effect of trap design, trap color, and a mixture of isothiocyanates on the capture of Ceutorhynchus assimilis (Paykull) was studied in a series of field experiments. Unbaited yellow water traps, a yellow sticky box trap, and a yellow sticky card trap, mounted vertically, or at 45° to the vertical with the yellow card facing upwards were effective for capturing seed weevils, but a horizontal yellow sticky card trap caught few. White, green, and black traps were unattractive. During migratory periods, trap catch could be enhanced two to four times with a lure consisting of a mixture of allyl, 3-butenyl, 4-pentenyl, and 2-phenylethyl isothiocyanates, but this bait did not attract in a trap with ineffective design. The sticky card trap, mounted at 45° to the vertical and baited with the isothiocyanate mixture, may be useful for monitoring movement of C. assimilis during migratory periods. However, during the colonization phase, lures of either a mixture of isothiocyanates or of allyl isothiocyanate alone were not attractive.

Field discrimination of oilseed rape, Brassica napus volatiles by cabbage seed weevil, Ceutorhynchus assimilis

The response of the cabbage seed weevil, Ceutorhynchus assimilis to yellow water traps baited with some components of oilseed rape (Brassica napus) odor was studied in a series of field experiments. Four isothiocyanates (NCS), five other amino acid derivatives (aromatic compounds), and two fatty acid derivatives are detected by peripheral olfactory receptors of C. assimilis. 3-Butenyl, 4-pentenyl, and 2-phenylethyl NCS were attractive when released individually during the period when weevils were immigrating to the host crop, but allyl NCS was not. A mixture of these four isothiocyanates was attractive during the periods of weevil immigration and dispersal. However, during the host colonization phase, 2-phenylethyl NCS, like the mixture of the four isothiocyanates, elicited no response or was repellent. Phenylacetonitrile and benzyl alcohol were attractive during the colonization phase. The fatty acid derivative (Z)-3-hexen-1-ol did not affect weevil catch in an experiment conducted when the insects were already established in the crop, but it depressed the catch in another conducted when weevils were dispersing from the crop. These results suggest that yellow traps baited with 2-phenylethyl NCS can be used to monitor immigration into crops in the spring and that phenylacetonitrile may be useful for assessing numbers of weevils colonizing the crop throughout the summer.

Male-produced aggregation pheromone in pea and bean weevil,Sitona lineatus (L.)

The attraction ofS. lineatus to live baits comprisingS. lineatus feeding onVicia faba (L.) was studied in a field experiment in the early spring. There was clear evidence that maleS. lineatus produced an aggregation pheromone which attracted approximately equal numbers of both sexes from overwintering sites. No evidence was obtained for the production, in the spring, of any semiochemical by female weevils.

Discrimination of oilseed rape volatiles by honey bee: Novel combined gas chromatographic-electrophysiological behavioral assay.

A novel technique for the simultaneous monitoring of electroan-tennogram (EAG) and conditioned proboscis extension (CPE) responses of honey bees to the effluent from a gas chromatograph (GC) was developed to locate biologically active components in blends of plant volatiles and to investigate odor recognition at the peripheral and behavioral levels. A six-component mixture, comprising compounds previously identified as oilseed rape floral volatiles, was used as the stimulus. Standard CPE and EAG recordings were done as a reference. EAG responses were elicited from unconditioned bees by all the components presented either in the coupled or the standard mode. Conditioned bees gave larger EAG responses than unconditioned bees, suggesting that antennal sensitivity is enhanced by conditioning. At the behavioral level, in both the standard and the coupled modes, only conditioned bees showed the proboscis extension response, with the majority of individuals responding to linalool, 2-phenylethanol, and benzyl alcohol.

Extraction, isolation, and composition of oviposition-deterring secretion of cabbage seed weevil Ceutorhynchus assimilis

Behavioral bioassays have confirmed that the oviposition-deterring secretion of the cabbage seed weevil, Ceutorhynchus assimilis Payk., can be isolated from glass tubes marked by the weevil and from extracts of its dissected seventh urotergite. Analysis of the secretion by gas chromatography—mass spectrometry showed that it contained iso- and n-alkanes, dimethylalkanes, alkenes, fatty acids, 15-nonacosanonc, 15-nonacosanol, and cholesterol. The oviposition-deterring properties of the secretion are associated with a more polar traction, isolated by liquid chromatography front an extract of the seventh urotergite.

Discrimination of oilseed rape volatiles by the honeybee: combined chemical and biological approaches

Honeybees (Apis mellifera L.) were individually subjected to a classical conditioning procedure in order to obtain an olfactory conditioned proboscis extension response. To relate the behavioural response directly to antennal detection abilities, a technique was developped for coupling proboscis extension responses and electroantennogram recordings, with the stimulation being provided by the effluent of a gas chromatograph (GC). Bees were conditioned with a six‐component mixture being part of oilseed rape (Brassica napus L.) floral volatiles, and tested with the individual components separated by GC. Responses of the conditioned bees were compared to those of unconditioned bees. No behavioural response was obtained in the control group, neither to the individual components nor to the mixture. Conditioning induced behavioural responses for three components, and an increase of electroantennogram responses for all components. A second experiment was conducted with an air entrainment extract of oilseed rape flower volatiles. Behavioural responses of conditioned and unconditioned bees were recorded. Responses obtained from conditioned bees tested with the air entrainment extract showed six groups of behaviourally active GC peaks. Unconditioned bees showed the same pattern of responses but at a lower level. The coupled technique described here appears to be a reliable tool for locating active components in a synthetic as well as in a natural mixture of floral volatiles. The effects of conditioning on odour discrimination and on its sensory correlates are discussed.