Transgenerational effects from single larval exposure to azadirachtin on life history and behavior traits of Drosophila melanogaster.

Azadirachtin is one of the successful botanical pesticides in agricultural use with a broad-spectrum insecticide activity, but its possible transgenerational effects have not been under much scrutiny. The effects of sublethal doses of azadirachtin on life-table traits and oviposition behaviour of a model organism in toxicological studies, D. melanogaster, were evaluated. The fecundity and oviposition preference of flies surviving to single azadirachtin-treated larvae of parental generation was adversely affected and resulted in the reduction of the number of eggs laid and increased aversion to this compound over two successive generations. In parental generation, early exposure to azadirachtin affects adult's development by reducing the number of organisms, delay larval and pupal development; male biased sex ratio and induced morphological alterations. Moreover, adult's survival of the two generations was significantly decreased as compared to the control. Therefore, Single preimaginal azadirachtin treatment can affect flies population dynamics via transgenerational reductions in survival and reproduction capacity as well as reinforcement of oviposition avoidance which can contribute as repellent strategies in integrated pest management programs. The transgenerational effects observed suggest a possible reduction both in application frequency and total amount of pesticide used, would help in reducing both control costs and possible ecotoxicological risks.

Identification of taste receptors and proteomic characterization of the antenna and legs of Tribolium brevicornis, a stored food product pest.

Chemoreception plays an important role in mediating a diverse range of behaviours, including predation and food selection. In the present study, we combined anatomical observations, electrophysiology and proteomics to investigate sensilla that mediate chemoreception on the antenna and the legs of Tribolium. Scanning electron microscopy was used to differentiate the coxal and trochanteral segments of the pro-, meso- and metathoracic legs by the presence of sensilla trichoidea and chaetica, while the antennae were covered with five types of sensilla (chaetica, basiconica, trichoidea, squamiformia and coeloconica). Antenna morphology and ultrastructure were similar in both sexes. Electrophysiological recordings allowed us to characterize a row of small sensilla basiconica on the terminal segment of the antenna as taste receptors, responding to sucrose and NaCl. Proteomics investigations of antennae and legs yielded several proteins with specific interest for those involved in chemoreception. Odorant-binding proteins were antenna-specific, while chemosensory proteins were detected in both tissues.

Two sugar isomers influence host plant acceptance by a cereal caterpillar pest.

Plant sugars are often considered as primary feeding stimuli, conditioning host plant acceptance by herbivorous insects. Of the nine sugars identified from methanolic extracts of seven grass species, only turanose, a sucrose isomer, was negatively correlated with the survival and growth of the noctuid larva of cereal stemborer, Busseola fusca. Sucrose was the most abundant sugar, although it did not vary significantly in concentration among the plant species studied. Using Styrofoam™ cylinders impregnated with increasing concentrations of turanose or sucrose, the two sugars had opposing effects: turanose appeared phagodeterrent while sucrose was phagostimulatory. Electrophysiological studies indicated that B. fusca larvae were able to detect both sugars via their styloconic sensilla located on the mouthparts. The findings indicate that, whereas sucrose is a feeding stimulant and positively influences food choice by B. fusca larvae, turanose negatively contributes to larval food choice. The balance in concentrations of both sugars, however, somehow influences the overall host plant choice made by the larvae. This can partly explain host plant suitability and choice by this caterpillar pest in the field.

Taste detection of phytoecdysteroids in larvae of Bombyx mori, Spodoptera littoralis and Ostrinia nubilalis.

A number of plants produce significant amounts of phytoecdysteroids that can disrupt the hormonal levels of insects feeding upon them. Insects equipped with taste receptors sensitive to phytoecdysteroids are able to avoid such plants. How common is this strategy? By recording from the lateral and medial sensilla styloconica in two polyphagous species (Ostrinia nubilalis and Spodoptera littoralis) and in a monophagous species (Bombyx mori), we tested whether the receptors could detect three commonly occurring phytoecdysteroids 20-hydroxyecdysone (20E), ponasterone A (ponA) and ecdysone (E). In B. mori, 20E and ponA elicited dose-dependent responses with a threshold of 1 &mgr;M only in the medial sensilla. In O. nubilalis, 20E, E and ponA elicited responses at threshold of 1 &mgr;M in both sensilla. In S. littoralis, 20E elicited responses with a threshold of 10 &mgr;M in both sensilla. By means of behavioural choice tests, we show that 20E is an effective feeding deterrent for O. nubilalis and S. littoralis first instar larvae. This suggests that the perception of phytoecdysteroids is more common among phytophagous lepidoptera than previously thought, although their toxicity or antifeedancy varies between species.

Biogenic amines modulate olfactory receptor neurons firing activity in Mamestra brassicae.

The modulatory effects of the biogenic amines octopamine and serotonin on pheromonal receptor neurons of Mamestra brassicae were investigated. The responses to sex pheromone components of two cells types (A and B) in single male long sensilla trichodea were monitored. Cell types A and B do not respond to the same compound. The response of type A to a pulse of the major sex pheromone component increased 5 min after octopamine injection. Responses of type B to other odorants increased after 30 min. In the absence of any pheromone stimulation the background firing activity of type A increased following octopamine injection. This background activity was used to evaluate the kinetics of octopamine and other biogenic amine effects on olfactory receptor neurons. Octopamine increased this background activity in a concentration- and time-dependent manner. Clonidine, an octopamine agonist, was shown to be more powerful in increasing the background activity of olfactory receptor neurons. The effects of octopamine and clonidine were hypothesized to arise from specific receptor activation as chlorpromazine (an octopamine antagonist) was shown to block the effect of octopamine. Serotonin, a known neuromodulator in most animal species, induced a reversible inhibition of spike firing. Altogether, these results indicate that biogenic amines can modulate the sensitivity of olfactory receptor neurons of moths either directly or by an action on adaptation.

Electrophysiological responses of female Helicoverpa armigera (Hübner) (Lepidoptera; Noctuidae) to synthetic host odours.

Studies were conducted to investigate the electroantennographic (EAG) responses of adult female Helicoverpa armigera to a range of known and putative kairomone components. The studies show that at a given dose the EAG responses elicited by a series of straight-chain aliphatic primary alcohols were not dependent on volatility since butan-1-ol and pentan-1-ol elicited EAG responses that were significantly smaller than those elicited by hexan-1-ol. The amplitudes of responses to hexan-1-ol were found to be dose dependent with a dose of 10(-1) µmol at source in a non-volatile solvent eliciting the largest response. Similarly, changes in functionality in a range of C(6) straight-chain aliphatic compounds significantly changed the amplitude of response elicited, with aldehydes eliciting smaller responses than the related primary alcohols and saturated compounds eliciting higher responses than related unsaturated compounds. Of the range of nine host plant-produced terpenoids tested, ocimene and beta-phellandrene elicited the highest responses and of the six aromatic compounds tested phenylacetaldehyde and benzaldehyde elicited the largest responses, at the doses tested. The significance of these findings for analysis of floral odours by gas chromatography linked to electroantennography as a means of identifying kairomone components attractive to H. armigera are discussed.

Sex-specific non-pheromonal taste receptors in Drosophila.

Taste receptors have recently been reported in Drosophila [1,2], but little is known of the relation between receptor and response. Morphological studies of the distribution of chemosensory sensilla indicate that the fruit fly has two major sites of gustation: the proboscis and the legs [3]. The taste sensilla on both these sites are similar in structure and each sensillum generally houses four gustatory neurons [4]. Early anatomical observations have demonstrated a sexual dimorphism in the number of tarsal sensilla [5] and in their central projections [6]. We measured the electrophysiological responses of the prothoracic taste sensilla to non-pheromonal substances--salts, sugars and water--and found a clear sexual dimorphism. From the response profile of individual sensilla, we were able to distinguish three types of tarsal sensilla in females as against only two types in males. The female-specific type, which responded specifically to sugar, was absent in males except when male gustatory neurons were genetically feminised. The fact that tarsal gustatory hairs exhibit a sexual dimorphism that affects the perception of non-pheromonal compounds suggests that sexual identity is more complex than has previously been thought [7,8].

Electrophysiological responses of gustatory sensilla of Mamestra brassicae (Lepidoptera, Noctuidae) larvae to three ecdysteroids: ecdysone, 20-hydroxyecdysone and ponasterone A.

Specialised phytophagous Lepidoptera such as Bombyx mori and Pieris brassicae have contact chemoreceptors that perceive ecdysteroids at very low concentrations. This sensory perception allows them to feed on substrates with a high content of phytoecdysteroids. We have evaluated if a polyphagous insect like Mamestra brassicae does possess contact chemoreceptor cells that are sensitive to these molecules. Electrophysiological recordings were performed from contact chemoreceptors located on the maxilla. These receptors were stimulated with some sugars, amino acids and salts and with three ecdysteroids. Our results demonstrate that a specific cell within the lateral sensilla responds to 20-hydroxyecdysone and ponasterone A but not to ecdysone.

A new method to improve olfactory responses to GC effluents.

Biological detectors such as the human nose or an insect antenna represent extremely attractive detectors for gas chromatography (GC) since they are specifically tuned to perceive biologically relevant compounds. However, these detectors are nonlinear, noisy and often perform poorly under standard GC conditions since they are adapted to detect transient rather than long-lasting stimuli. These drawbacks can be overcome if the chemical stimulus, delivered by the gas chromatograph, is re-shaped (modulated) before reaching the biological detector. We describe a pulsing system that improves the performance of an insect olfactory system when stimulated with the effluent from a GC capillary column. Chemicals eluted from the GC column are trapped and rapidly desorbed within a thermal modulator in order to transform the continuous effluent into a succession of short-pulsed stimuli. The output from this modulator was directed to an insect antenna, from which electrophysiological responses were recorded. The system was evaluated with adult male Spodoptera littoralis (Lepidoptera: Noctuidae) stimulated with the conspecific female sex pheromone. Results obtained with this new approach indicate that both sensitivity and reliability of the biological detector are improved compared with the classical technique. Possible developments of this new technique are discussed.

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.

Terpene variations in maritime pine constitutive oleoresin related to host tree selection byDioryctria sylvestrella RATZ. (Lepidoptera: Pyralidae).

Gas chromatographic analysis of the volatile terpenes of the wood oleoresin of maritime pines showed that the relative proportions of terpenes within the same tree did not vary according to sampling height and date. Pruning and tree vigor did not affect terpene profiles, but oleoresin composition varied widely from tree to tree. The trees attacked byDioryctria sylvestrella contained a significantly higher percentage of limonene, longipinene, and copaene, and a discriminant analysis, using the relative percentage of six terpenes, significantly separated infested and uninfested trees. The olfactory sensitivity of adult moths to a series of 20 volatile chemicals, including maritime pine oleoresin components, was evaluated with the electroantennogram method. Both sexes were sensitive to monoterpenes, oxygenated terpenoid compounds, and oleoresin samples. The use of terpenes as host attractants and biochemical markers for resistance toD. sylvestrella is discussed.

Object-oriented approach to fast display of electrophysiological data under MS-windows.

Microcomputers provide neuroscientists an alternative to a host of laboratory equipment to record and analyze electrophysiological data. Object-oriented programming tools bring an essential link between custom needs for data acquisition and analysis with general software packages. In this paper, we outline the layout of basic objects that display and manipulate electrophysiological data files. Visual inspection of the recordings is a basic requirement of any data analysis software. We present an approach that allows flexible and fast display of large data sets. This approach involves constructing an intermediate representation of the data in order to lower the number of actual points displayed while preserving the aspect of the data. The second group of objects is related to the management of lists of data files. Typical experiments designed to test the biological activity of pharmacological products include scores of files. Data manipulation and analysis are facilitated by creating multi-document objects that include the names of all experiment files. Implementation steps of both objects are described for an MS-Windows hosted application.

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.

Temporal coding of pheromone pulses and trains in Manduca sexta.

We investigated the ability of pheromone-sensitive olfactory receptors of male Manduca sexta to respond to 20-ms pulses of bombykal, the major component of the conspecific pheromonal blend. Isolated pulses of bombykal elicited a burst of activity which decreased exponentially with a time constant of 160-250 ms. Trains of pulses delivered at increasing frequencies (0.5-10 Hz) elicited temporally modulated responses at up to 3 Hz. Concentration of the stimulus (1, 10, 100 ng per odor source) had a marginal effect on the temporal resolution of the receptors. Within a train, the responses to individual pulses remained constant, except for 10-Hz trains (short-term adaptation). A dose-dependent decline of responsiveness was observed during experiments (long-term adaptation). Although individual neurons may not respond faithfully to each pulse of a train, the population of receptors sampled in this study appears to be capable of encoding the onset of odor pulses at frequencies of up to at least 3 Hz.

Sexual dimorphism of tarsal receptors and sensory equipment of the ovipositor in the European corn borer, Ostrinia nubilalis.

Sensilla on legs and ovipositor of the moth Ostrinia nubilalis were investigated by light and scanning electron microscopy. The ovipositor is composed of two papillae densely packed with medium length mechanoreceptor sensilla (MRb: 80-160 microns, n = 420-460). Long mechanoreceptor sensilla (MRa: 250-300 microns, n = 20-24) and contact chemoreceptors (CRa: 30-40 microns, n = 20-28) are evenly distributed at the periphery of these papillae. Legs support contact chemoreceptors (CRa), scattered among the scales. The pretarsus structure of each leg includes a single contact chemoreceptor (125 microns) inserted dorsally. The fifth tarsomere bears a ventral area without scales on which contact chemoreceptors are disposed in two parallel rows (CRb: 40-60 microns). A sexual dimorphism was found in the number and density of these sensilla (females: mean = 5.3, SD = 1.0; males: mean = 3.3, SD = 0.7), and in the size of the sensory field. The possible involvement of these sensory structures in oviposition site selection is discussed.

Floral volatiles ofTanacetum vulgare L. attractive toLobesia botrana den. et schiff. females.

The European grapevine moth (EGVM),Lobesia botrana, is a major pest of grapes in Europe. Females are attracted to a nonhost plant: tansy (Tanacetum vulgare L.), which is a common weed in Slovakian vineyards. A steam distillate extract of tansy flowers was analyzed by means of a GC-EAG technique to screen constituents detected by the olfactory receptors of EGVM females. From more than 200 GC peaks, nine peaks corresponding to monoterpenoids released an EAG response in more than 70% of the females (N=15):p-cymene,d-limonene,α-thujene,α-thujone,ß-thujone, thujyl alcohol, terpinene-4-ol, (Z)-verbenol, and piperitone. The steam distillate of tansy as well as a synthetic blend of identified compounds released consistent attraction in a field cage. The use of nonhost plants and host plant odors in integrated pest management is discussed.

Software filter for detecting spikes superimposed on a fluctuating baseline.

We describe and evaluate a software procedure for detecting and discriminating action potentials superimposed on a fluctuating DC baseline. Using an algorithm implemented in Fortran and Assembly language, spikes are detected by comparing the low-pass filtered first derivative of the signal with a preset threshold constant. This detection algorithm efficiently removes the DC baseline fluctuations usually encountered in sensory receptor recordings. It can function in real-time depending on the specific hardware configuration and program implementation. This approach enables the user to acquire direct DC-amplified signals, minimizing filter distortions of the action potential waveform.