Does Cry1Ab protein affect learning performances of the honey bee Apis mellifera L. (Hymenoptera, Apidae)?

Genetically modified Bt crops are increasingly used worldwide but side effects and especially sublethal effects on beneficial insects remain poorly studied. Honey bees are beneficial insects for natural and cultivated ecosystems through pollination. The goal of the present study was to assess potential effects of two concentrations of Cry1Ab protein (3 and 5000 ppb) on young adult honey bees. Following a complementary bioassay, our experiments evaluated effects of the Cry1Ab on three major life traits of young adult honey bees: (a) survival of honey bees during sub-chronic exposure to Cry1Ab, (b) feeding behaviour, and (c) learning performance at the time that honey bees become foragers. The latter effect was tested using the proboscis extension reflex (PER) procedure. The same effects were also tested using a chemical pesticide, imidacloprid, as positive reference. The tested concentrations of Cry1Ab protein did not cause lethal effects on honey bees. However, honey bee feeding behaviour was affected when exposed to the highest concentration of Cry1Ab protein, with honey bees taking longer to imbibe the contaminated syrup. Moreover, honey bees exposed to 5000 ppb of Cry1Ab had disturbed learning performances. Honey bees continued to respond to a conditioned odour even in the absence of a food reward. Our results show that transgenic crops expressing Cry1Ab protein at 5000 ppb may affect food consumption or learning processes and thereby may impact honey bee foraging efficiency. The implications of these results are discussed in terms of risks of transgenic Bt crops for honey bees.

Comparative sublethal toxicity of nine pesticides on olfactory learning performances of the honeybee Apis mellifera.

Using a conditioned proboscis extension response (PER) assay, honeybees (Apis mellifera L.) can be trained to associate an odor stimulus with a sucrose reward. Previous studies have shown that observations of conditioned PER were of interest for assessing the behavioral effects of pesticides on the honeybee. In the present study, the effects of sublethal concentrations of nine pesticides on learning performances of worker bees subjected to the PER assay were estimated and compared. Pesticides were tested at three concentrations. The highest concentration of each pesticide corresponded to the median lethal dose value (48-h oral LD50), received per bee and per day, divided by 20. Reduced learning performances were observed for bees surviving treatment with fipronil, deltamethrin, endosulfan, and prochloraz. A lack of behavioral effects after treatment with lambda-cyalothrin, cypermethrin, tau-fluvalinate, triazamate, and dimethoate was recorded. No-observed-effect concentrations (NOECs) for the conditioned PER were derived for the studied pesticides. Our study shows that the PER assay can be used for estimating sublethal effects of pesticides on bees. Furthermore, comparisons of sensitivity as well as the estimation of NOECs, useful for regulatory purposes, are possible.

Comparative toxicity and hazards of pesticides to Apis and non-Apis bees. A chemometrical study.

The adverse effects of 158 pesticides to the Honey bee (Apis mellifera), the alfalfa leafcutting bee (Megachile rotundata) and the alkali bee (Nomia melanderi) were compared by means of various linear and non-linear multivariate analyses. A comparison exercise including the bumble bee (Bombus spp.) was also performed from a more restricted set of 32 pesticides. While no difference of sensitivity was found between A. mellifera and Bombus spp., M. rotundata appeared the most susceptible to pesticides followed by N. melanderi.

Structure-toxicity modeling of pesticides to honey bees.

A quantitative structure-activity relationship (QSAR) model was derived for estimating the acute toxicity of pesticides on the honey bee. Chemicals were described by means of autocorrelation descriptors encoding lipophilicity (H), molar refractivity (MR) and the H-bonding acceptor ability (HBA) of the pesticides. A three-layer feedforward neural network trained by the back-propagation algorithm was used as statistical engine for deriving a powerful QSAR model. The root mean square residual (RMSR) values for the training and testing sets were 0.430 and 0.386, respectively. The practical interest of this original model was discussed.

Asymmetrical generalisation between pheromonal and floral odours in appetitive olfactory conditioning of the honey bee (Apis mellifera L.).

The capacity to generalise between similar but not identical olfactory stimuli is crucial for honey bees, allowing them to find rewarding food sources with varying volatile emissions. We studied bees' generalisation behaviour with odours having different biological values: typical floral odours or alarm compounds. Bees' behavioural and peripheral electrophysiological responses were investigated using a combined proboscis extension response conditioning-electroantennogram assay. Bees were conditioned to pure linalool (floral) or to pure isoamyl acetate (alarm) and were tested with different concentrations of both compounds. Electrophysiological responses were not influenced by conditioning, suggesting that the learning of individual compounds does not rely on modulations of peripheral sensitivity. Behaviourally, generalisation responses of bees conditioned to the alarm compound were much higher than those of bees conditioned to the floral odour. We further demonstrated such asymmetrical generalisation between alarm and floral odours by using differential conditioning procedures. Conditioning to alarm compounds (isoamyl acetate or 2-heptanone) consistently induced more generalisation than conditioning to floral compounds (linalool or phenylacetaldehyde). Interestingly, generalisation between the two alarm compounds, which are otherwise chemically different, was extremely high. These results are discussed in relation to the neural representation of compounds with different biological significance for bees.

Direct and indirect sublethal effects of Galanthus nivalis agglutinin (GNA) on the development of a potato-aphid parasitoid, Aphelinus abdominalis (Hymenoptera: Aphelinidae).

Snowdrop lectin (Galanthus nivalis agglutinin, GNA), has been shown to confer partial resistance to two potato aphids Myzus persicae and Aulacorthum solani, when incorporated in artificial diet and/or expressed in transgenic potato. First-tier laboratory-scale experiments were conducted to assess the potential effect of GNA on the aphid parasitoid Aphelinus abdominalis. GNA (0.1% w/v) was successfully delivered to Macrosiphum euphorbiae via artificial diet and induced a reduced growth rate and increased mortality compared to aphids fed a control diet. As aphid parasitoid larvae are endophagous, they may be exposed to GNA during their larval development and potential "chronic toxicity" on A. abdominalis was investigated. The amounts of GNA present in aphid and parasitoid tissues were estimated by western blotting. Results suggest that parasitoids excrete most of the GNA ingested. Sublethal effects of GNA on several parasitoid fitness parameters (parasitism success, parasitoid development and size, emergence success, progeny survival and sex ratio) were studied. No direct detrimental effect of GNA on A. abdominalis was observed. However, GNA had an indirect host-size-mediated effect on the sex ratio and the size of parasitoids developing in GNA-fed aphids. This work highlights the need to determine the exact "causes and effects" when assessing the ecological impact of transgenic plants on non-target beneficial insects. Such bioassays form the basis of a tiered risk assessment moving from laboratory studies assessing individuals towards field-scale experiments assessing populations.

Effects of artificial diet containing GNA and GNA-expressing potatoes on the development of the aphid parasitoid Aphidius ervi Haliday (Hymenoptera: Aphidiidae).

Aphid parasitoids are important biological control agents. The possibility arises that whilst foraging on insect-resistant transgenic plants, they are themselves at risk from direct and indirect effects of the expression of a transgene used to control the pest species. A liquid artificial diet was successfully used to deliver the snowdrop lectin (Galanthus nivalis agglutinin; GNA) to the peach-potato aphid, Myzus persicae. Bioassays utilising artificial diet incorporating GNA, and excised leaves of the GNA-expressing transgenic potato line, GNA2#28, were performed to assess the potential effects of GNA on the development of the aphid parasitoid Aphidius ervi. The results indicate that GNA delivered via artificial diet to the aphids can be transferred through the trophic levels and has a dose-dependent effect on parasitoid development. Parasitoid larvae excreted most of the ingested GNA in the meconium but some of it was detected in the pupae. Although A. ervi development was not affected when developing within hosts feeding on transgenic potato leaves, this probably reflected sub-optimal expression of the toxin in the transgenic potato line used

High level of resistance to proteinase inhibitors may be conferred by proteolytic cleavage in beetle larvae.

Incorporation of genes encoding proteinase inhibitors into oilseed rape genome could confer resistance to Coleoptera, which are the major pests on rape in Europe. A detailed study of the digestive proteinase of a model cruciferous-feeding Coleoptera, Phaedon cochleariae, showed that this insect relies on a complex proteolytic system including serine, cysteine, aspartyl proteinases, and leucine aminopeptidases. The inhibition of general and specific activities by a range of proteinase inhibitors in vitro suggested that oryzacystatin I (OCI) and Bowman-Birk inhibitor (BBI) would have adverse effects when ingested by the larvae. However, the growth and the feeding of larvae reared on oilseed rape leaf discs treated with a high dose of OCI and/or BBI were not affected. Moreover, the levels and patterns of proteolytic activities were not modified in these larvae. The study of the interactions between P. cochleariae larval proteinases and OCI and BBI revealed that both inhibitors were rapidly cleaved by serine proteinases in association with leucine aminopeptidases, and consequently lost their inhibitory capacity. This mechanism of resistance is very efficient, and may be widespread among Coleoptera. The major implications for insect control using proteinase inhibitor-based strategies are discussed.

Genetic variability of conditioned probing responses to a fruit odor in Leptopilina boulardi (Hymenoptera: Eucoilidae), a Drosophila parasitoid.

The genetic variability of odor-conditioned probing behavior was investigated in a population of Leptopilina boulardi, a parasitoid of Drosophila larvae. Ovipositor probing is the final step of host location, leading to the discovery of host larvae. It can be triggered by an odor previously experienced during an oviposition as a result of associative learning. This study was based on the analysis of female probing performance over two generations of isofemale lines (using both mother-daughter regressions and one-way analysis of variance). Individual performances of the conditioned response to the odor were characterized by (1) the latency (i.e., the time elapsed between the onset of the odor delivery and the start of the probing response), (2) the duration of the first probing phase, and (3) the total probing duration. Results suggested that the variability of two characters, the latency and the duration of the first probing phase, were under a genetic control in the studied population. This work is the first contribution to quantify the genetic component of this variability.

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.

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.

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 technique was used on restrained worker bees to study responses to components of an extract of oilseed rape floral volatiles. Bees were stimulated with the effluent from the gas chromatograph after either paired or unpaired conditioning to the extract, or after a control treatment. Proboscis extension activity was elicited in six areas of the chromatogram. However, the number of bees responding in two of these areas were too low to be considered in the present study. One significant area of activity was associated with the major component, (E,E)-alpha-farnesene, whilst the others were associated with several minor components. Although all three groups of bees, irrespective of the treatments applied, showed some responses to the components eluting from the GC column, only bees subjected to paired conditioning consistently responded when re-tested to the mixture. In addition, paired conditioning increased the responsiveness of individuals in terms of the number of bees responding at least once to the effluent from the gas chromatograph. This work confirmed the occurrence of key compounds in floral volatile mixtures. Possible synergistic/inhibitory effects between components, relating to olfactory experience, are discussed.

Olfactory learning and memory in the honeybee: comparison of different classical conditioning procedures of the proboscis extension response.

Olfactory learning in the honeybee was investigated using the conditioned proboscis extension reflex on restrained individuals. We compared, under the same experimental conditions, the most commonly used conditioning procedures, i.e. 1 trial, 3 massed trials (1 min inter-trial intervals), and 3 spaced trials (10 min inter-trial intervals) procedures, using linalool as the conditioned stimulus. Two experiments were performed in which worker bees were subjected to: (1) a single test at different times (30 s to 14 days) after the conditioning procedure; (2) a first test within 3 h after the conditioning procedure, and were then retested daily (up to 5 tests). The memory trace of a learnt odorant stimulus could last for the lifetime of the bee, even after a single association with sugar. Repeated tests with 1 day inter-test duration induced a strong decrease of the response level, this effect being more pronounced after a 1-trial conditioning.

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.

Computer analysis of the exploratory behavior of insects and mites in an olfactometer.

A method of quantification of the exploratory behavior of small animals stimulated by an odorant in a four-choice olfactometer, taking into account the interindividual variability of responses, was developed: individual tracks were time sampled according to the animal's walking speed and its positions were recorded according to the X-Y coordinates of the grid set underneath the device, the mesh of the grid suiting the animal's body size. A software, written in BASIC APPLESOFT on an APPLE IIe computer, allowed us to analyze the coordinates either of a single individual or of an experimental sample, leading to: a) the quantification of the insect distribution all over the experimental chamber, expressed in a table numbered according to the grid, where the percentage of position per square either for a given time fraction or the total observation period were reported, b) a graphic representation of the data according to several levels of greys, expressing the frequentation for each square for a given duration of observation. An analysis per time fraction allowed the chronological setup of events to appreciate. c) The collection of the positions among each flow field of the olfactometer for each individual of the experimental sample, for a given duration, was translated as the percentage of time spent in each flow field. Data files gathered these percentages for further statistical treatments. This computer method, which requires little equipment and appears to be easily adaptable to the study of biological models of various size and speed such as honeybees, trichogrammas and varroas mites, is a powerful tool for behavioral studies of small organisms tested in restricted areas.

Chemicals involved in honeybee-sunflower relationship.

We present a review of work on the plant chemicals involved in the honeybee-sunflower model system. Combined behavioral and chemical analyses were conducted under natural and controlled conditions. First the distribution of forager bees' visits on two pairs of sunflower genotypes producing a different level of hybrid seed yield was recorded under pollen-proof tunnels. Mirasol parental lines producing high seed yields were visited at random, whereas forager bees visited preferentially the female parental line of Marianne, resulting in low seed yield. Nectar samples collected on the genotypes were analyzed by gas chromatography. Fructose, glucose, and sucrose were identified. Parental lines of Mirasol showed similar sugar profiles, whereas the female line of Marianne contained higher amounts of sucrose than the male line. We assume that the bees' preferences between genotypes might rely on differences in the sugar composition of floral nectars, especially in the amount of sucrose. Aromas from headspace collection were compared between pairs and periodically during the flowering period. Of the 144 components indexed for Marianne lines and 136 components for Mirasol lines, 17 of the components for Marianne lines and 18 for Mirasol lines differed significantly according to flowering stage. Significant differences appeared in eight of the 134 components of Marianne lines and in 20 of the 250 components for Mirasol lines. Such differences, even restricted to a few components, might account for honeybees' discrimination between genotypes or flowering stage. Experiments then were conducted in a flight room using an artificial flower device. A total volatile extract was used as a conditioning scent previous to the test where the total extract was successively compared to several of its subfractions. Fractions significantly less visited than the total extract were discarded, whereas fractions confused with the total extract were kept. From step to step, a restricted fraction of 28 polar components, among which 15 were identified, was shown to be as active as the initial conditioning extract. These data emphasized honeybees' abilities to generalize from simplified to more complex chemical information. Finally, this work considers the possible use of such plant chemicals, from nectars or aromas, either as targets for genetic modification of crop plants or as direct attractants when sprayed on the crop, for the improvement of entomophilous cross pollination.

Sunflower aroma detection by the honeybee : Study by coupling gas chromatography and electroantennography.

Combined electrophysiological recordings (EAG) and gas chromatographic separation were performed in order to investigate which volatile chemical components of a sunflower extract could be detected by honeybee workers and thus are likely to trigger the foraging behavior. A direct coupling device allowed for the stimulation of the antennal receptors with individual constituents of a polar fraction of the flower aroma shown to be attractive to bees. More than 100 compounds were separated from the extract. Twenty-four compounds elicited clear EAG responses. These compounds were identified by mass spectrometry (electronic impact and chemical ionisation). Both short- and long-chain aliphatic alcohols, one short-chain aliphatic aldehyde, one acid, two esters, and terpenic compounds were found to stimulate the antennal receptors. Six compounds identified in previous behavioral experiments were found to exhibit EAG activity. The chemicals screened by this method may be used for recognition of the plant odor and the selective behavior of honeybees.

Sunflower volatiles involved in honeybee discrimination among genotypes and flowering stages.

In order to define the part of olfactory cues in the selective behavior of honeybees, observation on their foraging behavior was carried out on various sunflower genotypes in parallel with chemical analysis of aromatic extracts of the genotypes. Foragers show a preference for the early stages of flowering and, when they are given a choice between couples of parental lines of two commercial hybrids, Marianne and Mirasol, they are randomly distributed on Mirasol parents, but they prefer the female line of Marianne. The comparison of relative proportions of compounds among aromagrams obtained from head space trapping from the two couples of genotypes, reveals (1) a phenological stage effect for 17 compounds among 144 indexed compounds for Marianne lines and for 18 among 136 indexed compounds of Mirasol lines; most of these compounds exhibit higher relative proportions in the early flowering stages, which is related to plant attractiveness towards honeybees; (2) a sex effect for 33 compounds among 144 for Marianne lines and for 14 compounds among 136 for Mirasol lines; further semiquantitative analyses reveal a sex effect for only eight compounds of 134 for Marianne lines and 20 compounds of 250 for Mirasol lines, which represents less than 10% of the indexed compounds. These discriminatory compounds were partly identified by coupled GC-MS. Possible relations between such phenological and genotypical volatile fluctuations and forager attraction are discussed.

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.

Selective olfactory choices of the honeybee among sunflower aromas: A study by combined olfactory conditioning and chemical analysis.

A bioassay based on an olfactory conditioning method simulating the foraging situation in laboratory conditions was coupled with chemical analysis of volatile sunflower blends. Behavioural data obtained from for-agers'responses to volatile fraction point out that honeybees need to use only a limited fraction acting as a "simplified aromatic pattern" of the plant, among hundreds of compounds constituting the whole aroma. This active fraction included 27 polar compounds among which 14 were identified. Extension of such data to crop pollination and plant improvement is discussed.