Effect of Conditioning on Discrimination of Oilseed Rape Volatiles by the Honeybee: Use of a Combined Gas Chromatography-Proboscis Extension Behavioural Assay

The coupled gas chromatography-proboscis extension assay techniquewas used on restrained worker bees to study responses to componentsof an extract of oilseed rape floral volatiles. Bees were stimulatedwith the effluent from the gas chromatograph after either pairedor unpaired conditioning to the extract, or after a controltreatment. Proboscis extension activity was elicited in sixareas of the chromatogram. However, the number of bees respondingin two of these areas were too low to be considered in the presentstudy. One significant area of activity was associated withthe major component, (E, E)-     

Growth stimulation of beetle larvae reared on a transgenic oilseed rape expressing a cysteine proteinase inhibitor

The resistance of a transgenic line of oilseed rape expressing constitutively the cysteine proteinase inhibitor oryzacystatin I (OCI) was assessed against Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae). The levels of OCI expression in the transformed line averaged 0.2% and 0.05% of total soluble protein in leaves and petioles respectively. In vitro analyses showed that P. chrysocephala larvae use both cysteine and serine proteinases for protein digestion, and that all the cysteine proteolytic activity is OCI-sensitive. However, bioassays showed that adults fed identically on leaf discs from control or transformed plants. When larvae were reared on transgenic plants expressing OCI, they showed an increase in weight gain compared to those reared on control plants. Furthermore, those larvae from transgenic plants exhibited a 2-fold increase in both cysteine and serine proteolytic activity as a reponse to the presence of OCI. The plasticity of insect digestive physiology and feeding behaviour are discussed, as well as the relevance of engineering a genotype expressing both types of proteinase inhibitors.     

Molecular parameters involved in bee-plant relationships: a biological and chemical approach

Honeybee-plant relationships are based on a conditioning process in which olfactory (plant aroma) and gustatory cues (mainly nectars) are closely linked, leading to a selective foraging behaviour. Among crops dependent upon entomophilous cross-pollination, the sunflower has recently undergone extensive expansion due to hybrid variety selection. Sunflower hybrid seed production is strictly dependent upon pollinating insects, mainly the honeybees, but foragers may have preferences among the parental lines, leading to a lack of pollen carriage and consequently to a decrease of hybrid seed yield. In order to define the role of plant chemicals (aromas, nectars) involved in the pollination process, we set up a study combining behavioural and chemical assays. It appears that even though volatile chemical blends are much more complex compared to glucidic blends, for both kinds of cues only a 'limited chemical pattern' is responsible for foragers' choices. Therefore, it is henceforth possible to take these molecular criteria into account for plant improvement.     

Age dependency of worker bee response to queen pheromone in a four-armed olfactometer

Summary Behavioral responses of differently aged worker beesApis mellifera to a queen pheromonal extract were analysed. The bees were tested individually in a four-armed olfactometer, one arm being scented with the pheromonal extract. This extract was prepared from heads of 14–17-day-old unmated queens. Among the components of the blend, 470 g 9-keto-2-(E)-decenoic acid, 200 g 9-hydroxy-2-(E)-decenoic acid and 5 gp-hydroxybenzoic acid methyl ester per queen equivalent were dosed. An age dependency in the worker bees' olfactory response to the components of the queen extract was shown, the strongest response occurring below the age of 5 days.     

Two strains of cabbage seed weevil (Coleoptera: Curculionidae) exhibit differential susceptibility to a transgenic oilseed rape expressing oryzacystatin I

The aim of this study was to assess the potential effect of a transgenic line of oilseed rape expressing oryzacystatin I (OCI) on two strains of cabbage seed weevil. The level of OCI expression in seeds was approximately 0.05% of total soluble proteins. The insects were field-collected in two different locations, and their progeny was analyzed after a 3 week-development in pods. Both strains showed a similar pattern of proteolytic activity, and similar levels of OCI-sensitive proteinase activity in vitro. However, the larvae showed differential susceptibility to the transgenic plants. Despite inhibition of digestive proteinases in vitro by OCI in both strains, one strain showed an increased growth rate when fed the transgenic seeds, while the other strain remained unaffected. While suggesting the importance of studying individuals from different populations when assessing the effect of proteinase inhibitor-expressing plants on insect growth, our results also point out the necessity of studying the biochemical interactions taking place in vivo between the recombinant inhibitors and their target proteinases.     

Effects of Proteinase Inhibitor Ingestion on Survival,Learning Abilities and Digestive Proteinases of the Honeybee

The impact on beneficial insects of proteinase inhibitors expressed in pest-resistant transgenic crops needs to be assessed before the release of these plants into the environment. Three proteinase inhibitors, suitable for incorporation into oilseed rape, were tested on worker bees: the chicken egg white cystatin, oryzacystatin I (OCI) and Bowman-Birk soyabean inhibitor (BBI). Ingestion of low doses of the inhibitors did not cause short-term mortality, and a conditioned proboscis extension assay showed that olfactory learning performances were unchanged when the inhibitors were added to the reward. Long-term ingestion of BBI or OCI did not disrupt total digestive proteolytic activity, but ingestion of BBI induced a new proteinase form, suggesting the existence of a mechanism of control of proteinase synthesis in the honeybee.     

Olfactory responses ofTrichogramma maidis Pint,et Voeg.: Effects of chemical cues and behavioral plasticity

Oriented responses of Trichogramma maidisPint, et Voeg. to airborne odors were observed in a four-armed olfactometer. Experiments were carried out with odors of host eggs, the sex pheromone of Ostrinia nubilalisHbn, and maize extract, offered singly or in combination, both to naive wasps and to wasps previously exposed to the tested odor during an oviposition experience. The exploratory behavior in the olfactometer was quantified by means of a computer program which performed a space-time analysis of the insect 's movements. Whereas the naive wasps did not respond to the odor of the eggs, the synthetic sex pheromone of O. nubilalis,or the maize extract presented singly, they did react to a mixture of these three volatile cues. Prior oviposition in the odor of maize extract or in the combination of odors induced an increased preference toward the conditioning scent. This phenomenon did not occur when the wasps were conditioned to egg odor or sex pheromone alone. These results suggest that females can learn to associate some olfactory cues with the presence of the host. Immediately following the presentation of the combination of odors, a strong attraction of experienced wasps occurred; it decreased as the experiment progressed and finally reached the level presented by naive insects. Adult conditioning to the combination of odors also resulted in reduced variability in the behavioral responses.     

The attractiveness of different odour sources from the fruit–host complex on Leptopilina boulardi, a larval parasitoid of frugivorous Drosophila spp.

In parasitoid insects, successful offspring development depends on the female’s ability to find a suitable host. Specific recognition is often based on responses to olfactory cues, but their source and nature have rarely been determined. –This paper deals with the recognition of odours involved in host location by Leptopilina boulardi[Barbotin, Carton & Kelner-Pillault] (Hymenoptera: Eucoilidae), a larval parasitoid of Drosophila species that develops in mature fruits. The nature and origin of volatile stimuli recognized among odours of the host–fruit complex, and the effect of learning on this recognition, were investigated. Oriented responses to these odours were observed in a four-armed olfactometer and were analysed with the observer software (Noldus Information Technology). Fruit odours alone (banana and pear) were not spontaneously attractive to naive parasitoids, whereas naturally-infested bananas were highly attractive. The attraction was related to the odour that adult Drosophila left on the substrate but not to Drosophila oviposition activity or larval development. A synergism between some fruit odours (banana and pear) and the odour left by adult Drosophila on damp filter paper was observed. However, when testing a non-fruit substrate (mushroom), no synergism was observed. Thus, female L. boulardi may innately recognize host–food substrate odours associated with odours from the adult stage of their host. In addition, an oviposition experience on an infested banana allows L. boulardi females to memorise the fruit odour itself through associative learning. The adaptive significance of this process is discussed.     

Behavioural discrimination of oilseed rape volatiles by the honeybee Apis mellifera L.

This study investigated the ability of the honeybee to discriminatebetween six compounds previously identified as oilseed rapefloral volatiles: linalool, 2-phenylethanol, methyl salicylate,benzyl alcohol, (E)-2-hexenal and 1-octen-3-ol. These componentswere tested individually or in a synthetic mixture for theirability to elicit the conditioned proboscis extension response.Three experiments were done: conditioning to the mixture atone concentration (1.0, 0.1 or 0.01 µg per component)and testing to the individual components either at the conditioningor at the other concentrations; conditioning to individual componentsand testing to the mixture (1.0 µg); conditioning andtesting to the individual components (1.0 µg). The resultsfrom the proboscis extension assay were then compared to thoseobtained from free-flying bees in a flight room. From the conditionedproboscis extension assay, a conditioning threshold level wasfound (0.1 µg for the mixture studied) below which a reliableconditioning could not be achieved. Recognition thresholds wereobserved: bees responded to concentrations ten-fold higher orlower than that used for conditioning. Responses to lower concentrationswere weaker, whilst responses were increased at concentrationshigher than the conditioning one. A hierarchy within the compoundstested was found, with linalool, 2-phenylethanol and methylsalicylate cueing mixture recognition more effectively thanthe other components. The ranking order of the six componentswas similar in both the conditioned proboscis extension andthe conditioned foraging behaviour in flight room. When conditionedand tested to the individual components, bees discriminateda learned odour from a number of others. However, the specificitylevel for the recognition of the learned odour varied accordingto the component, the most clearly discriminated being the compoundswhich were used by the bee in mixture recognition.