Olfactory conditioning of proboscis activity in Drosophila melanogaster.

Olfactory learning and memory processes in Drosophila have been well investigated with aversive conditioning, but appetitive conditioning has rarely been documented. Here, we report for the first time individual olfactory conditioning of proboscis activity in restrained Drosophila melanogaster. The protocol was adapted from those developed for proboscis extension conditioning in the honeybee Apis mellifera. After establishing a scale of small proboscis movements necessary to characterize responses to olfactory stimulation, we applied Pavlovian conditioning, with five trials consisting of paired presentation of a banana odour and a sucrose reward. Drosophila showed conditioned proboscis activity to the odour, with a twofold increase of percentage of responses after the first trial. No change occurred in flies experiencing unpaired presentations of the stimuli, confirming an associative basis for this form of olfactory learning. The adenylyl cyclase mutant rutabaga did not exhibit learning in this paradigm. This protocol generated at least a short-term memory of 15 min, but no significant associative memory was detected at 1 h. We also showed that learning performance was dependent on food motivation, by comparing flies subjected to different starvation regimes.

Prior classical olfactory conditioning improves odour-cued flight orientation of honey bees in a wind tunnel.

Odours are key cues used by the honey bee in various situations. They play an important role in sexual attraction, social behaviour and location of profitable food sources. Here, we were interested in the role of odours in orientation at short distance, for instance the approach flight to a floral patch or in close proximity to the hive entrance. Using a newly designed wind tunnel, we investigated the orientation behaviour of the bee towards two different odours: a social odour and a floral component, linalool. We then tested the effect of prior olfactory conditioning (conditioning of the proboscis extension reflex) on subsequent flight orientation. We showed that both stimuli induced orientated behaviour (orientated flights, circling around the odour source) in up to 70% of the worker bees, social odour being slightly more attractive than the linalool. We found thereafter that orientation performance towards the floral compound can be significantly enhanced by prior classical olfactory learning. This type of information transfer, from a Pavlovian associative context to an orientation task, might allow future foragers to acquire, within the hive, relevant information about the odours and food they will encounter during their later foraging bouts.

Spontaneous recovery after extinction of the conditioned proboscis extension response in the honeybee.

In honeybees, the proboscis extension response (PER) can be conditioned by associating an odor stimulus (CS) to a sucrose reward (US). Conditioned responses to the CS, which are acquired by most bees after a single CS-US pairing, disappear after repeated unrewarded presentations of the CS, a process called extinction. Extinction is usually thought to be based either on (1) the disruption of the stored CS-US association, or (2) the formation of an inhibitory "CS-no US" association that is better retrieved than the initial CS-US association. The observation of spontaneous recovery, i.e., the reappearance of responses to the CS after time passes following extinction, is traditionally interpreted as a proof for the formation of a transient inhibitory association. To provide a better understanding of extinction in honeybees, we examined whether time intervals during training and extinction or the number of conditioning and extinction trials have an effect on the occurrence of spontaneous recovery. We found that spontaneous recovery mostly occurs when conditioning and testing took place in a massed fashion (1-min intertrial intervals). Moreover, spontaneous recovery depended on the time elapsed since extinction, 1 h being an optimum. Increasing the number of conditioning trials improved the spontaneous recovery level, whereas increasing the number of extinction trials reduced it. Lastly, we show that after single-trial conditioning, spontaneous recovery appears only once after extinction. These elements suggest that in honeybees extinction of the PER actually reflects the impairment of the CS-US association, but that depending on training parameters different memory substrates are affected.

Effects of sub-lethal and lethal doses of lambda-cyhalothrin on oviposition experience and host-searching behaviour of a parasitic wasp, Aphidius ervi.

In many parasitoid species, the recognition of chemical signals is essential to find specific hosts. This function is often impaired by exposure to insecticides that are usually neurotoxic. The behaviour of the Hymenopterous parasitoid Aphidius ervi (Haliday) (Hymenoptera: Aphidiinae) after surviving low doses of the pyrethroid lambda-cyhalothrin was examined in laboratory conditions. The host aphid was Myzus persicae (Sulzer) (Homoptera: Aphididae) on oilseed rape. Parasitoid females were exposed by contact with dry residues of the active ingredient at a lethal dose, LD20, and a sub-lethal dose, LD0.1. In a four-armed olfactometer, untreated and inexperienced females were attracted by the odour of M. persicae-infested plants and previous oviposition experience increased the duration of the attraction response. The response of inexperienced females decreased after an exposure to LD0.1 but not to LD20. No effect was observed when females had an oviposition experience prior to the olfactometer test. The oviposition activity was significantly decreased in the LD20-treated group but not in the LD0.1-treated one. All effects disappeared within 24h. Our work shows that orientation and oviposition behaviours may be impaired by low doses of lambda-cyhalothrin, depending on the dose, the parasitoid experience and the type of behaviour.

Effects of imidacloprid and deltamethrin on associative learning in honeybees under semi-field and laboratory conditions.

We have compared the sublethal effects of two insecticides in the honeybee (imidacloprid and deltamethrin) in both semi-field and laboratory conditions. A sugar solution containing 24 microg kg(-1) of imidacloprid or 500 microg kg(-1) of deltamethrin was offered to a colony set in an outdoor flight cage. In contrast to imidacloprid, deltamethrin had lethal effect on workers bees. The contamination of syrup with imidacloprid or deltamethrin induced a decrease in both the foraging activity on the food source and activity at the hive entrance. Negative effects of imidacloprid were also observed in an olfactory learnt discrimination task. Free-flying foragers were taken from the contaminated feeder and subjected to a conditioned proboscis extension response (PER) assay under laboratory conditions. As with free-flying bees, no impact of deltamethrin was found on the learning performances of restrained individuals in the PER procedure, whilst significant effects were found with imidacloprid in both semi-field and laboratory conditions.

Learning performances of honeybees (Apis mellifera L) are differentially affected by imidacloprid according to the season.

To establish the sublethal concentrations domain, acute and chronic oral tests were conducted on caged honeybee workers (Apis mellifera L) using imidacloprid and a metabolite, 5-OH-imidacloprid, under laboratory conditions. The latter showed a 48-h oral LD50 value (153 ng per bee) five times higher than that of imidacloprid (30 ng per bee). Chronic feeding tests indicated that the lowest observed effect concentrations (LOEC) of imidacloprid and of 5-OH-imidacloprid on mortality of winter bees were 24 and 120 microg kg(-1) respectively. Behavioural effects of imidacloprid and 5-OH-imidacloprid were studied using the olfactory conditioning of proboscis extension response at two periods of the year. Winter bees surviving chronic treatment with imidacloprid and 5-OH-imidacloprid had reduced learning performances. The LOEC of imidacloprid was lower in summer bees (12 microg kg(-1)) than in winter bees (48 microg kg(-1)), which points to a greater sensitivity of honeybees behaviour in summer bees, compared to winter bees.

Statistical analysis of honeybee survival after chronic exposure to insecticides.

Studies concerning long-term survival of honeybees raise the problem of the statistical analysis of mortality data. In the present study, we used a modeling approach of survival data of caged bees under chronic exposure to two pesticides (imidacloprid and deltamethrin). Our model, based on a Cox proportional hazard model, is not restricted to a specific hazard functional form, such as in parametric approaches, but takes into account multiple covariates. We consider not only the pesticide treatment but also a nuisance variable (variability between replicates). Moreover, considering the occurrence of social interactions, the model integrates the fact that bees do not die independently of each other. We demonstrate the chronic toxicity induced by imidacloprid and deltamethrin. Our results also underline the role of the replicate effect, the density-dependent effect, and their interactions with the treatment effect. None of these parameters can be neglected in the assessment of chronic toxicity of pesticides to the honeybee.